CN111295196A

CN111295196A - Molecular bacterial therapy for controlling skin enzyme activity

Info

Publication number: CN111295196A
Application number: CN201880065645.XA
Authority: CN
Inventors: R·L·伽罗; M·威廉姆斯
Original assignee: University of California
Current assignee: University of California
Priority date: 2017-08-31
Filing date: 2018-08-31
Publication date: 2020-06-16
Also published as: JP7373176B2; BR112020003508A2; JP2024009974A; EP3675884A4; CA3072772A1; KR20200083435A; JP2020536494A; US20210177917A1; WO2019046801A1; MX2020002013A; EP3675884A1; AU2018326791B2; AU2018326791A1

Abstract

The present invention provides a purified polypeptide that inhibits (i) protease production and/or activity of keratinocytes, (ii) IL-6 production and/or activity of keratinocytes, (iii) Staphylococcus aureus production of phenol soluble regulatory protein α 3, and/or (iv) AGR production and/or activity of Staphylococcus aureus.

Description

Molecular bacterial therapy for controlling skin enzyme activity

Statement regarding federally sponsored research

The present invention was made with government support granted by the National Institutes of Health under grant numbers AI117673, AR067547, AR062496, and AR 064781. The government has certain rights in this invention.

Cross Reference to Related Applications

According to 35 article 119 of the american codex, the disclosure of which is incorporated herein by reference, this application claims priority to provisional application serial No. 62/553,025 filed on 31/8/2017.

Technical Field

The present disclosure relates to compositions and methods for treating dermatological diseases and conditions, and to compositions for modulating skin barrier permeability.

Preservation of microorganisms

Exemplary microorganisms of the present disclosure [ Staphylococcus epidermidis (Staphylococcus epidermidis) a11, Staphylococcus Hominis (Staphylococcus Hominis) C5, Staphylococcus Hominis A9 and Staphylococcus waderi (Staphylococcus swarneri) G2] were deposited as ATCC No. _______ (strain name Staphylococcus epidermidis a 1181618, deposited at 28.8.2018) from American Type Culture Collection of university of massachas university at 10801 No. va.20110-2209 under the budapest treaty about 2018.28, ATCC No. _______ (strain name Staphylococcus Hominis C1618, deposited at 28.8.2018), ATCC No. _______ (strain name Staphylococcus epidermidis a981618, deposited at 28.8.8.8), and ATCC No. _______ (strain name Staphylococcus waderidis G2818, deposited at _______ (strain wo 2818, wo 2818). The deposit will be maintained at the authorized depository for the longest period of time, for a period of at least five years since the latest sample publication request was received at the depository, for a period of at least thirty years since the date of deposit, or during the relevant patent expiration (energetically valid), and will be replaced in the event of a mutation, inactivity or disruption. All of the limitations to public access to these cell lines are irreversibly removed after the patent issuing from this application.

Background

The epidermis is the first line of immune defense, which protects and regulates the interaction between microorganisms and host organisms. Controlling this interaction is very important because the bacteria not only reside on the surface where they affect superficial keratinocytes, but also penetrate down to the stratum corneum and into the dermis where some bacterial species have been shown to affect immune function. For example, staphylococcus epidermidis (s. epidermidis) interacts with epidermal keratinocytes to prevent toll-like receptor 3-mediated inflammation, recruit mast cells and T cells, and increase tight junctions (light junctions) and antimicrobial peptide production. Unlike the common skin commensal bacterium, staphylococcus epidermidis, staphylococcus aureus (s. This is particularly evident in skin diseases such as Atopic Dermatitis (AD) promoted by staphylococcus aureus.

It has been demonstrated that the overall microbial diversity of the microbiome resident in the skin of subjects with AD is reduced and that the abundance of staphylococcus aureus is increased. Increased colonization by staphylococcus aureus has been associated with increased disease severity in AD patients. Mechanistically, it is not clear how staphylococcus aureus worsens the disease. Several products from staphylococcus aureus have been shown to disrupt the barrier and/or initiate inflammation. These products include alpha toxin, superantigen, toxic shock syndrome toxin 1, enterotoxin, protein A, Panton-Valentine leukocidin, exfoliative toxin, and V8 serine protease. Because of the potentially pathogenic role of these molecules, understanding the skin's response to staphylococcus aureus colonization in the absence of clear signs of clinical infection is crucial to understanding the pathogenesis of AD and developing future therapies.

Disclosure of Invention

The present disclosure provides a purified polypeptide comprising a sequence having at least 98% identity to SEQ ID No. 4, 11, 12, 13, 14, 15, 16, or 17 and inhibiting (i) protease production and/or activity of keratinocytes, (ii) IL-6 production and/or activity of keratinocytes, (iii) inhibiting the production of phenol soluble regulatory protein α by s.aureus (s.aureus) and/or (iv) inhibiting the production and/or activity of agr by s.aureus.

The present disclosure also provides topical formulations comprising a polypeptide of the present disclosure or a compound of formula I, IA or IB.

The present disclosure also provides an isolated polynucleotide encoding a polypeptide of the disclosure. In one embodiment, the polynucleotide comprises a sequence that hybridizes under stringent conditions to a polynucleotide consisting of SEQ ID NO. 1 or 3 and encodes a polypeptide comprising SEQ ID NO. 4. In further embodiments, the polynucleotide comprises SEQ ID NO 1 or 3.

The disclosure also provides vectors comprising the polynucleotides of the disclosure. The vector may be any suitable vector for expression in a cellular or microbial host.

The present disclosure also provides a recombinant microorganism comprising a vector or polynucleotide of the present disclosure. In some embodiments, a microorganism that does not naturally express a polypeptide of the disclosure is engineered to express a polynucleotide of the disclosure by recombinant engineering. In yet further embodiments, the microorganism is attenuated in that it has become non-pathogenic or reduced pathogenic as compared to a wild-type organism of the same species. In yet further embodiments, the recombinant microorganism is a microorganism (e.g., a commensal microorganism) typically found on the skin of a mammal (e.g., a human).

The present disclosure also provides a probiotic composition comprising a recombinant microorganism of the present disclosure.

The present disclosure also provides a probiotic composition comprising a microorganism expressing a polypeptide of the present disclosure (e.g., SEQ ID NOs: 4, 11, 12, 13, 14, 15, 16, and/or 17). In one embodiment, the microorganism is staphylococcus hominis, staphylococcus epidermidis, staphylococcus wovensis, or any combination thereof. In a further embodiment, the microorganism is staphylococcus hominis C5, staphylococcus hominis a9, staphylococcus epidermidis a11 and/or staphylococcus wavorans G2. In still further or further embodiments, the composition comprises a microorganism selected from the group of microorganisms consisting of: the microorganism has ATCC No. _______ (strain name staphylococcus epidermidis a 1181618, deposited 28 months at 2018), ATCC No. _______ (strain name staphylococcus epidermidis C581618, deposited 28 months at 2018 months 28 days at 2018), ATCC No. _______ (strain name staphylococcus epidermidis a981618, deposited 28 months at 2018 months 28 days at 2018), ATCC No. _______ (strain name staphylococcus wadskii G281618, deposited 28 months at 2018 months 8 days) and combinations of any of the foregoing. In additional embodiments, the probiotic compositions of the present disclosure are non-natural (e.g., do not include all of the microorganisms found on the skin, or include an amount per unit volume of microorganisms not found on the skin, or the microorganisms have been genetically modified, or the compositions contain ingredients or compounds not normally found on the skin).

The present disclosure also provides a method of treating a dermatological disorder comprising administering an effective amount of a coagulase-negative Staphylococcus species (cos sp.) or an effective amount of a fermented extract of cos sufficient to inhibit protease activity on skin, wherein the cos produces a polypeptide comprising a sequence having at least 98% identity to SEQ ID No. 4, 11, 12, 13, 14, 15, 16, or 17 and inhibits protease production. In one embodiment, the dermatological disorder is selected from the group consisting of: netherton syndrome, atopic dermatitis, contact dermatitis, eczema, psoriasis, acne, epidermal hyperkeratosis, acanthosis, epidermal inflammation, dermal inflammation and pruritus. In further embodiments, the administering is by topical application. In still further or further embodiments, the cos is selected from the group consisting of: staphylococcus epidermidis, Staphylococcus capitis (Staphylococcus capitis), Staphylococcus caprae (Staphylococcus caprae), Staphylococcus saccharolyticus (Staphylococcus saccharolyticus), Staphylococcus wowen, Staphylococcus pasteuri (Staphylococcus pasteurii), Staphylococcus haemolyticus (Staphylococcus hyicus), Staphylococcus delbrueckii (Staphylococcus devriesii), Staphylococcus hominis, Staphylococcus aureus, Staphylococcus jettensis, Staphylococcus petasisi, and Staphylococcus lugdongensis (Staphylococcus lugdongensis). In still another or further embodiment of any of the preceding embodiments, the fermented extract of CoNS comprises the peptide sequence of SEQ ID NO. 4 and/or the compound of formula I, IA or IB. In further embodiments, the CoNS is selected from the group consisting of: staphylococcus epidermidis a11, staphylococcus hominis C4, staphylococcus hominis C5, staphylococcus hominis a9, staphylococcus woolli G2 and any combination thereof.

The present disclosure also provides a method of treating a skin disease or disorder, comprising measuring protease activity of a culture from the skin of a subject or protease activity of the skin of a subject; comparing protease activity to a normal control; applying a symbiotic skin bacteria composition and/or a fermented extract from coagulase-negative staphylococci, wherein the symbiotic skin bacteria composition or fermented extract comprises a polypeptide having at least 98% identity to

SEQ ID NO

4, 11, 12, 13, 14, 15, 16 or 17 and/or comprises a compound of formula I, IA or IB, wherein the composition is formulated as a cream, ointment or pharmaceutical composition that maintains the growth and replication capacity of symbiotic skin bacteria. In one embodiment, the coagulase-negative staphylococcus is selected from the group consisting of: staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus saccharolyticus, Staphylococcus wowensis, Staphylococcus pasteuri, Staphylococcus haemolyticus, Staphylococcus delbrueckii, Staphylococcus hominis, Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus lugdunensis.

The present disclosure also provides a method of treating a skin disease or disorder comprising administering a purified polypeptide or probiotic composition of the present disclosure, the probiotic composition comprising a polypeptide-producing bacterium having at least 98% identity to

SEQ ID NO

4, 11, 12, 13, 14, 15, 16, or 17 and inhibiting the production or activity of kallikrein.

The present disclosure also provides a method of treating a skin disease or disorder comprising administering a composition that inhibits expression of a phenol soluble regulatory protein, wherein the composition comprises a purified polypeptide of the present disclosure or a compound of formula I, IA or IB. In one embodiment, the administration is topical administration. In another embodiment, the composition is a fermented extract of coagulase-negative staphylococci.

The present disclosure also provides a topical probiotic composition comprising probiotic commensal skin bacteria selected from the group consisting of: staphylococcus epidermidis a11, staphylococcus hominis C4, staphylococcus hominis C5, staphylococcus hominis a9, staphylococcus woolli G2 and any combination thereof. In one embodiment, the composition is formulated as a lotion, a shake, a cream, an ointment, a gel, a foam, a powder, a solid, a paste, or a tincture.

The present disclosure also provides a pharmaceutical composition comprising a drug and a staphylococcus aureus fermented extract containing the phenol soluble regulatory protein α 3 or a staphylococcus aureus probiotic.

The present disclosure provides symbiotic/beneficial bacteria and/or products thereof to prevent an increase in protease activity in the skin. This is important in many disease states, including atopic dermatitis, netherton syndrome, and other skin conditions that suffer from abnormally high protease activity and barrier disruption.

The present disclosure also provides factors and compositions to induce protease activity to aid in the proteolytic remodeling of skin in the treatment of conditions associated with wound repair, aging, sunburn, pigmentary abnormalities, and scarring.

The present disclosure provides a method of treating a dermatological condition comprising administering an effective amount of coagulase-negative staphylococcus species (cos) or an effective amount of a fermented extract of cos sufficient to inhibit protease activity on skin. In one embodiment, the dermatological disorder is selected from the group consisting of: netherton syndrome, atopic dermatitis, contact dermatitis, eczema, psoriasis, acne, epidermal hyperkeratosis, acanthosis, epidermal inflammation, dermal inflammation and pruritus. In further embodiments, the administration is by topical application. In further embodiments, the CoNS is selected from the group consisting of: staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus saccharolyticus, Staphylococcus wowensis, Staphylococcus pasteuri, Staphylococcus haemolyticus, Staphylococcus delbrueckii, Staphylococcus hominis, Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus lugdunensis. In a specific embodiment, the CoNS is Staphylococcus epidermidis.

The present disclosure also provides a method of treating a skin disease or disorder, comprising measuring protease activity from a culture of the skin of a subject or protease activity from the skin of a subject; comparing protease activity to a normal control; applying a symbiotic skin bacteria composition and/or a fermented extract from coagulase-negative staphylococci, wherein the symbiotic skin bacteria composition comprises at least one symbiotic bacteria that reduces serine protease activity of the culture or skin, wherein the at least one symbiotic bacteria is formulated as a cream, ointment or pharmaceutical composition that maintains the growth and replication capacity of the symbiotic skin bacteria. In one embodiment, the coagulase-negative staphylococcus is selected from the group consisting of: staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus saccharolyticus, Staphylococcus wowensis, Staphylococcus pasteuri, Staphylococcus haemolyticus, Staphylococcus delbrueckii, Staphylococcus hominis, Staphylococcus aureus jettentis, Staphylococcus aureus, and Staphylococcus lugdunensis.

The present disclosure also provides a method of treating a skin disease or disorder comprising administering an agent that inhibits the expression of kallikrein. The present disclosure also provides a method of treating a skin disease or disorder comprising administering an agent that inhibits expression of a phenol soluble regulatory protein. In one embodiment of any of the preceding embodiments, said administering is topical administering. In further embodiments, the agent is a fermented extract of coagulase-negative staphylococci. In further embodiments, the coagulase-negative staphylococcus is selected from the group consisting of: staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus saccharolyticus, Staphylococcus wowensis, Staphylococcus pasteuri, Staphylococcus haemolyticus, Staphylococcus delbrueckii, Staphylococcus hominis, Staphylococcus aureus jettentis, Staphylococcus petasii and Staphylococcus lugdunensis.

The present disclosure also provides topical compositions comprising a plurality of skin bacteria. In one embodiment, the probiotic commensal skin bacteria are coagulase-negative staphylococcus species. In a different embodiment, the probiotic commensal skin bacteria comprise staphylococcus aureus. In one embodiment of any of the preceding embodiments, the bacteria are formulated as a cream, lotion, tincture, gel or other topical formulation (formulary) in which the bacteria remain viable.

The present disclosure also provides a topical probiotic composition comprising a probiotic commensal skin bacteria fermented extract obtained from coagulase-negative staphylococcus (CoNS) species. In one embodiment, the CoNS is selected from the group consisting of: staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus saccharolyticus, Staphylococcus wowensis, Staphylococcus pasteuri, Staphylococcus haemolyticus, Staphylococcus delbrueckii, Staphylococcus hominis, Staphylococcus aureus jettentis, Staphylococcus petasii and Staphylococcus lugdunensis.

In any of the embodiments described, the topical probiotic composition is formulated as a lotion, a shake, a cream, an ointment, a gel, a foam, a powder, a solid, a paste, or a tincture.

The present disclosure provides a pharmaceutical composition comprising a drug and a staphylococcus aureus fermentation extract or a staphylococcus aureus biological composition.

The present disclosure provides a method for drug delivery through the skin comprising contacting the skin with a composition comprising a drug and a staphylococcus aureus fermentation extract or a staphylococcus aureus biologic composition. In one embodiment, the drug is a topical drug to be absorbed or adsorbed through the skin.

The present disclosure also provides a method of delivering a topical drug comprising contacting the skin of a subject with a composition comprising staphylococcus aureus or a fermented extract of staphylococcus aureus for a period of time and at a dose and under conditions to increase the permeability of the skin, and then contacting the skin with the drug to be delivered.

The present disclosure provides a composition comprising a fermented extract from staphylococcus aureus or a lotion, shake, cream, ointment, gel, foam, powder, solid, paste, or tincture containing live staphylococcus aureus.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

Drawings

FIGS. 1A-D show that (A-C) NHEK was treated with sterile filtered supernatants of Staphylococcus aureus (SA; Newman, USA300, 113, SANGER252) and Staphylococcus epidermidis (ATCC12228, ATCC1457) for 24 hours and NHEK conditioned media was analyzed with specific trypsin-like, elastase-like, and MMP protease substrates. (D) The effect of proteases secreted by Staphylococcus aureus (Newman) on trypsin activity was analyzed. Data represent mean ± SEM (n ═ 4) and represent at least three independent experiments. One-way analysis of variance (ANOVA) (aec) and two-way analysis of variance (d) were used and significance was indicated by P <0.05, P <0.001, P < 0.0001. ANOVA, analysis of variance; MMP, matrix metalloproteinase; NHEK, normal human epidermal keratinocytes.

FIGS. 2A-C show that (A) total protease activity was measured in NHEK conditioned media (5 μ g ml BODIPY FL casein) after 0-48 hours of Staphylococcus aureus (SA, Newman) supernatant treatment; (B) however, serpin aprotinin (800. mu.g ml) was applied to the conditioned medium 24 hours after treatment; (C) the effect of Staphylococcus aureus (USA300 LAC) WT and protease-free (protease-null) strains on NHEK conditioned medium trypsin activity (Boc-Val-Pro-Arg-AMC, 200mM) was compared. Two-way analysis of variance (A, B) and one-way analysis of variance (C) were used and significance was indicated by P <0.05, P <0.01, P <0.001, P < 0.0001. ANOVA, analysis of variance; NHEK, normal human epidermal keratinocytes; WT, wild type.

FIGS. 3A-F show that Staphylococcus aureus increases KLK expression in human keratinocytes. (A) The relative abundance of KLK mRNA expression in NHEK was analyzed by qPCR after 24h staphylococcus aureus (SA, Newman) supernatant treatment. (B-E) analysis of fold-changes in mRNA expression of KLK5, 6, 13 and 14 in NHEK treated with Staphylococcus aureus supernatant for 0-48 hours. All mRNA expression levels were normalized by the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (F) Using both published and predicted molecular weights, NHEK conditioned media and cell lysates were analyzed by immunoblotting for changes in protein expression of KLK5, 6, 13 and 14 after 24 hours of treatment with sa (newman) supernatant. The housekeeping gene, a-tubulin, was used as a loading control for cell lysates. Data represent mean ± SEM (n ═ 3) and represent at least three independent experiments. Two-way analysis of variance was used (bee), and significance was indicated by P <0.01, P <0.001, P < 0.0001. ANOVA, analysis of variance; KLK, kallikrein; NHEK, normal human epidermal keratinocytes; qPCR, real-time quantitative PCR; SEM, standard error of mean.

FIGS. 4A-D show that multiple KLKs cause Staphylococcus aureus-induced serine protease activity in human keratinocytes. In CaCl₂Prior to differentiation and addition of staphylococcus aureus (Newman) supernatant, NHEK was treated with KLK6, 13 or 14siRNA (15 nM). siRNA scrambled (scrambled) (-) controls 1 and 2 were used at 15nM and 45nM, respectively. (A) Conditioned media were analyzed for changes in trypsin activity (Boc-Val-Pro-Arg-AMC, 200. mu.M). (B-D) the transcript levels of KLK6, KLK13 and KLK14 were assessed by qPCR and normalized to the housekeeping gene GAPDH to confirm siRNA knockdown efficiency. Data represent mean ± SEM (n ═ 4) and represent at least three independent experiments. Analysis of (a) using one-way variance, and by P<0.05、**P<0.01、***P<0.001 indicates significance. ANOVA, analysis of variance; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; KLK, kallikrein; NHEK, normal human epidermal keratinocytes; qPCR, real-time quantitative PCR; siRNA, small interfering RNA; SEM, standard error of mean.

FIGS. 5A-C show that multiple KLKs modulate Staphylococcus aureus-induced DSG-1 and FLG lysis in human keratinocytes. Changes in NHEK after siRNA knockdown of KLK6, 13 and 14(15nM) were assessed by immunoblotting for 24 hours of treatment with staphylococcus aureus (Newman) supernatant as follows: (A) desmoglein-1 (DSG-1) and (B) profilaggrin (Pro-FLG) are cleaved. The housekeeping gene a-tubulin was used as a loading control. DSG-1 (full length) and Pro-FLG are indicated by black arrows. (C) Densitometric analysis of both DSG-1 (full length) and Pro-FLG is expressed by the average number of pixels normalized to a-tubulin (n ═ 1). Immunoblots represent at least three independent experiments. KLK, kallikrein; NHEK, normal human epidermal keratinocytes; siRNA, small interfering RNA.

FIG. 6 depicts a method of preparing a fermentation extract and performing an activity assay.

FIG. 7 shows that under the control of agr quorum sensing system (quorum sensing system), Staphylococcus aureus phenol soluble regulatory Protein (PSM) is responsible for the increased keratinocyte serine protease activity.

FIG. 8 shows that Staphylococcus aureus PSM increases mouse serine protease activity and skin barrier damage.

FIG. 9 shows that S.aureus isolates from Atopic Dermatitis (AD) damaged skin can induce serine protease activity in keratinocytes in an agr-type dependent manner.

FIG. 10 shows that coagulase-negative Staphylococcus (CoNS) strain ATCC14490 (Staphylococcus epidermidis) can produce an autoinducing peptide (AIP) to turn off the agr activity of Staphylococcus aureus.

FIG. 11 shows the effect of Staphylococcus aureus and commensal bacteria on serine protease activity in atopic dermatitis.

FIG. 12 shows the effect of human Staphylococcus aureus C5 on the agr activity of Staphylococcus aureus.

FIG. 13 shows the effect of various CoNS strains on the agr activity of Staphylococcus aureus.

FIGS. 14A-J show that Staphylococcus aureus PSM α causes disruption of epithelial barrier homeostasis.A human keratinocyte (NHEK) was stimulated with sterile-filtered supernatant of Staphylococcus Aureus (SA) from wild-type (WT), PSM α (Δ PSM α) or PSM β (Δ PSM β) knock-out strains for 24h and analyzed for (A) trypsin activity and (B) KLK6mRNA (n 4) compared to the housekeeping gene GAPDH (C) PSM synthetic peptide was added to NHEK for up to 24h to analyze the change in trypsin activity (D, E) to evaluate transcriptional analysis of genes that changed by more than or equal to 2-fold after PSM α 3 treatment by RNA-Seq followed by Gene Ontology (GO) analysis.A protease knock-out strain (Δ protease) (1 e) secreted by SA WT, SA Δ PSM α or SA 10) (1 e-1 e)⁷CFU) 8-week-old male C57BL/6 mice (n ═ 6) were treated for 72 h. (F, G) representative picture of murine skin (dotted line indicates treated area) and change in thickness of epidermis after treatment (scale bar 200 μm). (H-K) also evaluation of the skin on the back of mice (WT or mutant SA strains) for transepidermal water loss (TEWL) and SA CFU/cm²A change in (c). All error bars are expressed as Standard Error of Mean (SEM) and statistical significance indicated by the following was determined using one-way analysis of variance: p is a radical of<0.05*、p<0.01**、p<0.001***、p<0.0001****。

FIGS. 15A-G show that Staphylococcus epidermidis agr type I self-induced peptides are characteristic of and deficient in AD skin. (A, B)24h inhibition of staphylococcus aureus agr type I-III supernatant on Staphylococcus Aureus (SA) USA300LAC agr type I activity (n ═ 4), and a representation of the known structure of staphylococcus epidermidis agr type I autoinducing peptides (AIPs). (C) Effect of staphylococcus epidermidis (s.epi) agr type I strain RP62A Wild Type (WT) or self-induced peptide knock-out (Δ AIP) on SA agr activity after 24 h. (D) Sterile-filtered supernatant or Δ AIP supernatant of SA grown in the presence or absence of staphylococcus epidermidis WT was applied to NHEK for another 24h, and then NHEK trypsin activity was measured (n ═ 4). (E) Consensus of the staphylococcus epidermidis agr type I-III genome found on AD skin. (F, G) ratio of Staphylococcus epidermidis agr type I to SA relative abundance on the ruber region of 8 individual AD subjects based on the 'least severe' to 'most severe' AD score based on objective SCORAD and the combined data for all subjects based on AD severity. All error bars are expressed as Standard Error of Mean (SEM) and used one-way analysis of variance (A, C, D) and (nonparametric) unpaired Mann-Whitney test (F) to determine statistical significance indicated by: p <0.05, p <0.01, p <0.001, p < 0.0001.

FIGS. 16A-F show that multiple clinically isolated strains of coagulase-negative Staphylococcus inhibit Staphylococcus aureus agr activity. (A) A sterile filtered supernatant of clinically isolated coagulase-negative staphylococci (bons) was added to Staphylococcus Aureus (SA) USA300LAC agr type I P3-YFP reporter strain (reporter strain) for 24h, and then SA agr activity was analyzed (n ═ 3). (B, C) the genome of human Staphylococcus C5 strain was further sequenced, and the sequence of the auto-inducible peptide (AIP) of the agrD gene was analyzed. Biochemical analysis of human staphylococcus C5 supernatant the ability of <3kDa size exclusion centrifugation, 80% ammonium sulfate precipitation and pH 111 h treated supernatant to affect SA agr activity was also tested. (D-F) SA grown in the presence of supernatant of human Staphylococcus C5 for 24h was sterile filtered and added to human keratinocytes (NHEK) for 24h, then analyzed for trypsin activity, KLK6mRNA expression compared to the housekeeping gene GAPDH, and IL-6 protein levels. All error bars are expressed as Standard Error of Mean (SEM) and a one-way analysis of variance was used to determine statistical significance indicated by: p <0.05, p <0.01, p <0.001, p < 0.0001.

FIGS. 17A-H show that clinical CoNS isolate of AD inhibits SA-induced murine skin barrier damage. In the Presence or absence of active human Staphylococcus aureus C5(1 e)⁸CFU), Staphylococcus Aureus (SA) USA300LAC agr type I pAmiP3-Lux reporter strain (1 e)⁷CFU) were applied to 8-week-old female C57BL/6 mice for 48h (n-5). (A, B) evaluation of SA agr activity on the skin of the back of the mice by luminescence change. (C) Representative image of murine skin after 48h SA treatment (dashed boxes indicate treatment areas). (D-H) determination of SA CFU/cm²And murine skin barrier injury and inflammation were assessed by analyzing Il 6mRNA expression, trans-dermal water loss (TEWL), trypsin activity, and changes in Klk6mRNA expression normalized to the housekeeping gene Gapdh. All error bars are expressed as Standard Error of Mean (SEM) and a one-way analysis of variance was used to determine statistical significance indicated by: p is a radical of<0.05*、p<0.01**、p<0.001***、p<0.0001****。

Figures 18A-H show that staphylococcus aureus PSM α altered major barrier genes and cytokine expression in human keratinocytes, (a-D) the change in trypsin activity and KLK6 transcriptional expression normalized to the housekeeping gene GAPDH were evaluated in both a dose and time dependent manner for human keratinocytes treated with synthetic PSM α 3, (E) GO-term analysis of genes that were downregulated by >2 fold relative to control in human keratinocytes treated with PSM α 3 24H, (F-H) the change in human keratinocyte cytokine protein expression of IL-6, TNF- α, or IL-1 α treated with SA WT, SA Δ PSM α, or SA Δ PSM β supernatant for 24H, all error bars are expressed as mean Standard Errors (SEM), and a one-way variance analysis was used to determine statistical significance as indicated by p <0.05, p <0.01, p <0.001, p < 0.0001.

FIGS. 19A-H show that Staphylococcus aureus PSM α and protease are responsible for barrier damage and inflammation induction on mouse skin Staphylococcus Aureus (SA) (1 e)⁷CFU) Wild Type (WT), PSM α knock-out (Δ psm α) and protease-free (Δ proteinase) strains were applied to male rat dorsal skin for 72h (n ═ 6) and changes in IL17a/f mRNA expression normalized to the housekeeping gene Gapdh were measured (A, E) for trypsin activity, (B, F) Klk6, (C, G) Il6 and (D, H). all error bars are expressed as Standard Errors of Mean (SEM) and one-way anova was used to determine statistical significance as indicated by p<0.05*、p<0.01**、p<0.001***、p<0.0001****。

FIGS. 20A-C show that CoNS strains did not affect SA growth. Coagulase-negative staphylococcal (CoNS) supernatant affected the growth of SAagr type I P3-YFP reporter strain as assessed by OD600nm (n ═ 3-4), including (A) CoNS clinical isolates, (B) Staphylococcus epidermidis agr type I-III and (C) Staphylococcus epidermidis wild-type (WT) or self-induced peptide knockout (Δ AIP) supernatants added to the SA agr type I reporter strain for 24 h. All error bars are expressed as standard error of the mean (SEM).

FIGS. 21A-B show that human Staphylococcus aureus C5 inhibited SA agr type I-III, but not type IV. Human staphylococcal C5 supernatant was added to SA agr type I-IV P3-YFP reporter strain for 24h (n ═ 3). (A) SA reporter strain agr type I-IV activity and (B) measurement of the growth of OD600nm when cultured in the presence of supernatant of Staphylococcus hominis C5. All error bars are expressed as Standard Error of Mean (SEM) and a one-way analysis of variance was used to determine statistical significance indicated by: p <0.05, p <0.01, p <0.001, p < 0.0001.

FIGS. 22A-F show that human Staphylococcus C5 supernatant inhibits SA-induced skin barrier damage. Concentrated in the presence or absence of 10 ×)<Staphylococcus Aureus (SA) (1 e) in the case of 3kDa human Staphylococcus C5 supernatant⁷CFU) was applied to the dorsal skin of female rats for 48h (n-3). (A-B) representative image of the back of the rat (dotted line indicates treatment area) and SA CFU/cm recovered from the skin of the rat after SA treatment². (C-F) SA-induced skin barrier damage markers including Il6, trans-dermal water loss (TEWL), trypsin activity and Klk6mRNA expression compared to the housekeeping gene Gapdh. All error bars are expressed as standard error of the mean (SEM) and are assignedSingle factor analysis of variance was used to determine statistical significance indicated by: p is a radical of<0.05*、p<0.01**、p<0.001***、p<0.0001****。

Detailed Description

As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an agent" includes a plurality of such agents, and reference to "the microorganism" includes reference to one or more microorganisms and equivalents thereof known to those skilled in the art, and so forth.

In addition, the use of "or" means "and/or" unless stated otherwise. Similarly, "comprise," comprises, "includes" and "includes" are interchangeable and not intended to be limiting.

It will be further understood that where the description of various embodiments uses the term "comprising," those skilled in the art will understand that in some specific cases, the language "consisting essentially of …" or "consisting of …" may alternatively be used to describe embodiments.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Any methods and agents similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions.

Atopic Dermatitis (AD) is one of the most common immune diseases, placing a heavy burden on the quality of life and financial status of patients and bringing a serious risk of complications, defects in skin barrier function are an important feature of AD, elevated levels of Th2 cytokines (e.g., IL4 and IL13) of eczematous skin lesions in AD patients Th2 cytokines promote a decrease in skin barrier function by inhibiting expression of filaggrin, these cytokines also inhibit expression of human antimicrobial peptides such as antimicrobial peptides (cathelicidins) and b-defensin-2, which are defects in AD that can lead to dysbiosis of the skin bacteria community and enhanced colonization of staphylococcus aureus, therapy targeting IL4 receptors α leads to significant improvement of the disease Th2 strong correlation between cytokine activity, barrier function, antimicrobial activity and disease outcome supports efforts to determine causal relationships between these basic epidermal functions.

Increased proteolytic activity can compromise the skin barrier of AD patients, as they have been found to exhibit increased kallikrein (KLK) expression. KLK is a family of 15 serine proteases, several of which are mainly present in the upper granular and corneal layers of the epidermis. In Netherton syndrome, an increase in serine protease activity is observed due to a decrease in the activity of the serine protease inhibitor, Kazal-5 type. The resulting increase in enzyme activity results in increased desquamation, altered antimicrobial peptide and Filaggrin (FLG) processing, and protease activated receptor 2 activation and inflammation. Increased protease activity may also play an important role in the communication of the microbiome with the skin immune system, and it has recently been shown to directly affect epidermal cytokine production and inflammation by enhancing penetration of bacteria through the epidermis.

Dysbiosis of the skin microflora and colonization of staphylococcus aureus on the skin are associated with the worsening of Atopic Dermatitis (AD). The present disclosure demonstrates that staphylococcus aureus has the ability to induce the expression of specific KLK from keratinocytes and increase the overall proteolytic activity in skin. This illustrates the system by which bacteria on the skin communicate with the host and presents a previously unknown but potentially important mechanism for how colonization by staphylococcus aureus will increase disease severity in AD patients.

Staphylococcus aureus can secrete a variety of proteases onto the skin, which alter the integrity of the skin barrier. Serine protease V8 and serine-like protease epidermal exfoliating toxins have been shown to cleave corneodesmosomal (corneodesmosome) adhesion proteins, including DSG-1, resulting in increased desquamation. Aureolysin (Aureolysin), an MMP, is known to cleave and inactivate LL-37, an important antimicrobial peptide on the skin. However, these direct proteolysis of S.aureus products requires high levels of enzymes and bacteria and is therefore more consistent with events that occur during infection by this organism.

Increased digestion of barrier proteins was observed after s.aureus activated keratinocytes. FLG is known to be cleaved from the larger Pro-FLG (400kDa) into a monomeric form (37kDa) that plays an important role in forming the physical barrier of the stratum corneum and keratin. Accelerated cleavage of Pro-FLG has been shown to be associated with increased skin desquamation (Hewett et al, 2005). Interestingly, increased lysis of Pro-FLG was observed in human keratinocytes treated with Staphylococcus aureus supernatant. Cleavage of Pro-FLG was partially blocked when KLK6 or KLK13 were silenced, indicating that staphylococcus aureus can reduce the integrity of the skin barrier in a KLK-dependent manner by cleaving Pro-FLG.

DSG-1 is an important keratinocyte desmosomal adhesion protein that, when cleaved, results in increased desquamation. Full-length DSG-1(160kDa) in keratinocytes was readily cleaved by s.aureus-stimulated KLK activity. It was reported that KLK5, 6, 7 and 14 can cleave DSG-1, whereas KLK13 cannot. This suggests that upregulated KLK6 and KLK14 may lead to enhanced cleavage of full-length DSG-1 while providing evidence contrary to the view that KLK13 does not participate in DSG-1 cleavage. Thus, s.aureus can cause KLK to alter FLG lysis, but also increase DSG-1 lysis as another way to reduce epidermal skin barrier integrity. Specific siRNA knockdown indicates that an increase in KLK expression is at least partially responsible for the increased serine protease activity stimulated by s. Figure 2C shows that secreted proteases from staphylococcus aureus contribute to inducing an increase in trypsin activity in keratinocytes. Bacteria including staphylococcus aureus may also affect the protease activity of skin cells because they can penetrate the skin surface and cause a strong skin immune response (Nakatsuji et al, 2013, 2016; Zhang et al, 2015). These observations are also associated with rosacea or netherton syndrome.

The present disclosure demonstrates that soluble factors produced by staphylococcus aureus have an effective, previously unexpected ability to alter the endogenous protease activity produced by keratinocytes. This occurs in dilutions of the S.aureus product from which no activity of the bacterial protease could be detected. Thus, staphylococcus aureus can promote the epidermal enhancement of the expression of endogenous proteolytic activity, thereby significantly altering the balance of total epidermal proteolytic activity.

Different strains of staphylococcus aureus (Newman, USA300, 113 and SANGER252) and staphylococcus epidermidis (ATCC12228 and ATCC1457) have different effects on human keratinase activity. Strains of staphylococcus aureus (including Newman and USA300) increased trypsin activity, while other strains of staphylococcus aureus and staphylococcus epidermidis increased elastase or MMP activity. Thus, bacteria can alter epidermal protease activity, depending on the species and strain of the bacteria. Other bacterial species and strains of staphylococcus aureus may additionally uniquely affect the enzymatic balance of human skin. Interestingly, preliminary data found that purified toll-like receptor ligands did not induce trypsin activity or KLK expression in keratinocytes.

Protease activity is highly upregulated in a variety of skin diseases, leading to impaired skin barrier. In almost all cases, this is associated with a worsening disease condition. In one aspect, the present disclosure demonstrates that symbiotic microorganisms and their bacterial products can be used to prevent an increase in protease activity in skin. In particular, the present disclosure demonstrates that coagulase-negative staphylococci can prevent s.aureus-induced serine protease activity in skin by inhibiting the agr quorum sensing system. Staphylococcus aureus is a pathogenic bacterial strain that induces serine protease activity in the skin. Increased protease activity disrupts the skin barrier and leads to exacerbated disease states including netherton syndrome and atopic dermatitis. The present disclosure demonstrates that this increase in serine protease activity can be prevented by using commensal or benign skin bacteria and factors derived therefrom.

The present disclosure addresses the unexpected response of keratinocytes to staphylococcus aureus. As a result of increased DSG-1 and FLG lysis, staphylococcus aureus produces one or more factors that reduce the integrity of the skin barrier in a KLK-dependent manner.

The present disclosure demonstrates that S.aureus secretes a phenolic soluble regulatory protein α (PSM α), which triggers autodigestion of the epidermis.

The present disclosure also identifies commensal bacteria, genes and polypeptides that inhibit the accessory gene regulator (agr) quorum sensing system of staphylococcus aureus and turn off PSM α, thereby inhibiting protease activity.

The present disclosure demonstrates that coagulase-negative staphylococcal (CoNS) species that normally reside on the skin, such as Staphylococcus epidermidis and Staphylococcus hominis, are protected from this biological activity of Staphylococcus aureus by the production of auto-inducing peptides (AIP) that inhibit the accessory gene regulated (agr) quorum sensing system of Staphylococcus aureus and turn off PSM α secretion.

Almost all staphylococcus aureus toxins are under the control of a virulence accessory gene regulator (agr). The agr system triggers changes in gene expression at specific cell densities through a process called quorum sensing. In addition to toxins, agr is known to up-regulate various virulence determinants, such as extracellular enzymes (proteases, lipases, nucleases), and down-regulate the expression of surface-bound proteins. This adaptation is thought to control the production of certain virulence determinants of the infection when needed (e.g., binding proteins when cell density is low at the beginning and adhesion to host tissues is important); and controlling the production of toxins and degrading extracellular enzymes when an infection is established and nutrition needs to be taken from the host tissue.

Various clinical isolates of different CoNS species inhibited protease activation and prevented epithelial damage both in vitro and in vivo without altering the abundance of Staphylococcus aureus (e.g., inhibited protease bioactivity/agr activity without altering Staphylococcus aureus density). Furthermore, the present disclosure demonstrates that patients with active AD show a reduction in the relative abundance of these beneficial microorganisms (e.g., cos) compared to staphylococcus aureus, thus overcoming the inhibition of quorum sensing and enabling staphylococcus aureus to break the barrier. In summary, the present disclosure shows how members of the normal human skin microbiota maintain immune homeostasis by acting as a community to help control staphylococcus aureus toxin production.

The present disclosure also identifies polynucleotide sequences, polypeptide sequences, and fragments thereof that provide products that inhibit agr quorum sensing activity. These polynucleotides and polypeptides are useful for providing therapeutic agents and recombinant non-pathogenic skin bacteria or attenuated skin bacteria for use in topical formulations to treat staphylococcus aureus infections and/or atopic dermatitis.

For example, the present disclosure provides an autoinducing peptide (AIP) that down-regulates agr activity. Also provided herein are polynucleotides encoding AIPs.

The present disclosure provides a link between increased colonization of staphylococcus aureus in AD skin and increased serine protease activity, and provides novel targets and therapies, including but not limited to: a fermented extract that upregulates protease activity in skin (e.g., a fermented extract from staphylococcus aureus); or a fermented extract from commensal bacteria that down-regulates protease activity in the skin (e.g., containing one or more AIPs of the disclosure). In addition, the present disclosure provides (i) topical formulations comprising such extracts or purified AIP peptides; (ii) a topical formulation comprising commensal probiotic bacteria (e.g., non-pathogenic or attenuated bacteria that have been transformed with an AIP coding sequence, or a purified commensal bacterial preparation in a topical formulation). Additional therapeutic targets may be antibodies directed to KLK and/or DSG-1 and/or FLG therapies (e.g., to increase expression of these factors or delivery to AD subjects).

In one embodiment, the AIP polypeptides of the disclosure have X₁X₂X₃X₄CX₅X₆X₇X₈(SEQ ID NO:10) wherein X is₁Is S, K, V, G or T; x₂Is Y, Q, A orI；X₃Is N, S, T or D; x₄Is V, P, M or T; x₅Is G, S, A, N or T; x₆Is G, N, T or L; x₇Is Y or F; and X₈Is F, L or Y, wherein amino acids 5-9 of SEQ ID NO 10 form a thiolactone ring. Exemplary peptide sequences that fall within the consensus sequence of SEQ ID NO 10 include: SYNVCGGYF (SEQ ID NO:4), KYNPCSNYL (SEQ ID NO:11), SYSPCATYF (SEQ ID NO:12), SQTVCSGYF (SEQ ID NO:13), GANPCALYY (SEQ ID NO:14), TINTCGGYF (SEQ ID NO:15), VQDMCNGYF (SEQ ID NO:16) and GYSPCTNFF (SEQ ID NO: 17). In a further embodiment, the polypeptide yields a structure of formula I or IA. In another embodiment, the polypeptide may comprise a combination of D-amino acids or L-amino acids. In any of the preceding embodiments, the polypeptide inhibits staphylococcus aureus protease activity, agr activity, or keratinocyte protease activity.

The present disclosure provides compounds of formula I:

wherein X₁From 1-6 amino acids; x₂Is an amino acid selected from the group consisting of: valine (V), proline (P), methionine (M), and threonine (T); wherein R is⁵Selected from the group consisting of:

wherein R is⁶Selected from the group consisting of:

wherein R is⁷Selected from the group consisting of:

and wherein R⁸Selected from the group consisting of:

in one embodiment, the present disclosure provides a compound of formula IA:

wherein X₁From 1-6 amino acids; x₂Is an amino acid selected from the group consisting of: valine (V), proline (P), methionine (M), and threonine (T);

wherein R is¹Selected from the group consisting of:

wherein R is²Selected from the group consisting of:

wherein R is³Selected from the group consisting of:

wherein R is⁵Selected from the group consisting of:

wherein R is⁶Selected from the group consisting of:

wherein R is⁷Selected from the group consisting of:

and wherein R⁸Selected from the group consisting of:

the present disclosure provides purified polypeptides (e.g., AIP peptides) comprising a sequence having at least 98% identity to SEQ ID NO:4 and which inhibit (i) protease production and/or protease activity of keratinocytes, (ii) IL-6 production and/or activity of keratinocytes, (iii) production of phenol soluble regulatory protein α 3 by staphylococcus aureus (s. aureus), and/or (iv) agr production and/or activity of staphylococcus aureus.

In yet further embodiments, the present disclosure provides a purified polypeptide comprising or consisting of SEQ ID No. 4, 11, 12, 13, 14, 15, 16 or 17. In further embodiments, the polypeptide forms a structure of formula I, IA or IB.

In one embodiment, AIP peptides of the present disclosure may comprise one or more D-amino acids.

The present disclosure provides topical formulations comprising an AIP peptide having the consensus sequence of SEQ ID No. 10, or a peptide of SEQ ID No. 4, 11, 12, 13, 14, 15, 16 or 17, or a compound of formula I, IA or IB.

By "substantially identical" is meant that the amino acid sequences are largely (but not completely) identical, but retain the functional activity of the sequences with which they are associated. The percent identity shared with a polypeptide sequence or a polynucleotide sequence is based on the alignment of the sequences. It is common in the art to perform alignments and determine identity using a variety of programs. Generally, two polypeptides or domains are "substantially identical" if their sequences have at least 85%, 90%, 95%, 98%, or 99% identity, or if there are conservative variations in the sequences. Sequence identity can be compared using computer programs, such as the BLAST program (Altschul et al, 1990).

The disclosure also provides polynucleotides encoding the AIP polypeptides of the disclosure (i.e., "AIP polynucleotides"). For example, the present disclosure provides polynucleotides encoding SEQ ID NO. 2 or 4. In one embodiment, the polynucleotide hybridizes under stringent conditions to a polynucleotide consisting of SEQ ID NO. 3 and encodes a polypeptide of SEQ ID NO. 4. The "stringency" of the hybridization reaction is readily determinable by one of ordinary skill in the art, and is typically an empirical calculation dependent on probe length, washing temperature, and salt concentration. Generally, longer probes require higher temperatures for proper annealing, while shorter probes require lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of homology desired between the probe and hybridizable sequence, the higher the relative temperature that can be used. Thus, as a result, higher relative temperatures tend to make the reaction conditions more stringent, while lower temperatures are less so. For more details and explanations on the stringency of hybridization reactions, see Ausubel et al, Current Protocols in Molecular Biology, Wiley Interscience Publishers (1995). As defined herein, "stringent conditions" or "highly stringent conditions" typically: (1) washing with low ionic strength and high temperature, e.g. 0.015M sodium chloride/0.0015M sodium citrate/0.1% sodium lauryl sulfate at 50 ℃; (2) denaturing agents such as formamide, e.g., 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer pH 6.5 at 42 ℃, and 750mM sodium chloride, 75mM sodium citrate; or (3) at 42 ℃ using 50% formamide, 5 XSSC (0.75M NaCl, 0.075M sodium citrate), 50mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 XDenhardt's solution, sonicated salmon sperm DNA (50. mu.g/ml), 0.1% SDS, and 10% dextran sulfate, with washing at 42 ℃ in 0.2XSSC (sodium chloride/sodium citrate) and at 55 ℃ in 50% formamide, followed by high stringency washing at 55 ℃ (consisting of 0.1 XSSC with EDTA). The codon table can be used to derive polynucleotide sequences encoding

SEQ ID NOs

11, 12, 13, 14, 15, 16 and 17.

The AIP polynucleotide may be cloned into various vectors for use in the present disclosure, for example, the AIP polynucleotide may be cloned into an expression vector or plasmid for transformation and/or expression in recombinant host cells, the vectors for bacterial transformation are known, the four major types of vectors are plasmids, viral vectors, cosmids and artificial chromosomes, the aiP polynucleotide may be inserted into a clone, vector, shuttle, plasmid, BAC, or may also be integrated into the bacterial genome if a plasmid is used, the copy number of the plasmid may be between 5 and 500 copies per cell, exemplary plasmids and expression vectors include, but are not limited to, p252, p256, p353-2(Leer et al, 1992), p 4-2, pApApApApACYC, pUJJ-derivatives (ujccic & Topisivic, 1987, etc., strains, etc., and strains, Lip, 1987, Lip, 1987, Lip, 2000, Lip, rice.

For example, in one embodiment, the topical composition comprises a purified polypeptide (e.g., an AIP peptide) comprising the consensus sequence of SEQ ID NO:10 or a sequence having at least 98% identity to any of

SEQ ID NOs

4, 11, 12, 13, 14, 15, 16, or 17, and inhibits (i) protease production and/or protease activity of keratinocytes, (ii) inhibits IL-6 production and/or activity of keratinocytes, (iii) inhibits the production of phenol soluble regulatory protein α from staphylococcus aureus (s. aureus), and/or (iv) inhibits agr production and/or activity of staphylococcus aureus.

In additional embodiments, the topical composition may comprise a non-pathogenic microorganism (including attenuated microorganisms that have been engineered to reduce or eliminate pathogenic activity), wherein the microorganism has been engineered to express an AIP polypeptide. The microorganism can be engineered to contain a vector and/or AIP polynucleotide. In one embodiment, the microorganism produces a compound of formula I, IA and/or IB.

In one embodiment, the compositions and methods herein use a non-pathogenic bacterium that has been engineered by transforming the bacterium with an AIP polynucleotide of the present disclosure, thereby producing a compound of formula I, IA and/or IB. In one embodiment, the bacteria in the population are non-pathogenic, non-invasive microorganisms, and in certain embodiments may be gram-positive food grade bacterial strains. In additional embodiments, the transformed bacterial population is prepared from bacteria naturally occurring in the skin microbiome.

In certain embodiments, the bacteria forming the bacterial population in the composition and transformed with the compounds of expression I, IA and/or IB may be a collection of the same bacteria or a mixture of different bacteria at different phylogenetic levels. Bacteria residing on healthy human skin include bacterial species that normally reside on the human face, such as bacteria in the actinomycetes (Actinobacteria), including the corynebacterium (corynebacterium) and the propionibacterium (propionibacterium). In other embodiments, the bacteria residing on the skin of a healthy human subject include bacterial species that are typically present on skin other than the face, including bacteria in genera such as bacteroidetes (bacteroidetes) and proteobacteria (proteobacteria). Other bacteria in the skin microbiota lineage include those listed herein below.

In one embodiment, the bacteria are from the genus propionibacterium, including but not limited to: propionibacterium acidiproducens (Propionibacterium acidififaciens), Propionibacterium acidiproducens (Propionibacterium acidipropionici), Propionibacterium acidipropionici strain 4900, Propionibacterium acnes (Propionibacterium acnes), Propionibacterium camphorata (Propionibacterium australiensis), Propionibacterium avariensis (Propionibacterium avium), Propionibacterium cyclobutylicum (Propionibacterium cyclaxacum), Propionibacterium freudenreichii (Propionibacterium freudenreichii), Propionibacterium freudenreichii subsp 71, Propionibacterium freudenreichii subsp. capable of being transformed into a bacterium, Propionibacterium freudenreichii strain 02, Propionibacterium freudenreichii strain 4977, Propionibacterium acidipropionici strain (Propionibacterium jensenii), Propionibacterium freudenreichii strain 02, Propionibacterium freudenreichii (Propionibacterium), Propionibacterium jejunipeniponicum frenulatum strain (Propionibacterium) and Propionibacterium jejunipenii (Propionibacterium). In one embodiment, the bacterium is not propionibacterium acnes. In one embodiment, the bacteria are from the genus corynebacterium, including but not limited to: corynebacterium crowding (c.acolensis), corynebacterium non-fermentum (c.aerophilum), corynebacterium amycolatum (c.amycolatum), corynebacterium argentatum (c.argentonatense), corynebacterium aquaticum (c.aquaticum), corynebacterium otobacter (c.auris), corynebacterium bovis (c.bovis), corynebacterium diphtheriae (c.diphtheria), corynebacterium equi (c.equi) [ rhodococcus equi (rhodococcus equi) ], corynebacterium flavum (c.flavescens), corynebacterium gluconicum (c.glucoronella), corynebacterium glutamicum (c.glutamicum), corynebacterium granulosum (c.grandinosucum), corynebacterium haemolyticum (c.haemaceum), corynebacterium hayrolyticum, corynebacterium jeikeium (c.jeikeium) group k, corynebacterium maytans (c.maculophyllobacterium), corynebacterium pseudorhizobium sheep (c.corynebacterium flavipes), corynebacterium pseudorhizobium Corynebacterium urealyticum (c.urealyticum) (group D2), corynebacterium renosum (c.renale), corynebacterium species (c.spec), corynebacterium striatum (c.striatum), corynebacterium tenuis (c.tenuis), corynebacterium ulcerans (c.ulcerans), corynebacterium urealyticum and corynebacterium sicum (c.xerosins). Bacteria having lipophilic groups and non-lipophilic groups are contemplated, and non-lipophilic bacteria may include fermentative corynebacteria and non-fermentative corynebacteria. In one embodiment, the bacterium is not corynebacterium diphtheriae, c. amicolatum, corynebacterium striatum, corynebacterium jeikeium, corynebacterium urealyticum, corynebacterium xerosis, corynebacterium pseudotuberculosis, corynebacterium tenuis, corynebacterium striatum, or corynebacterium mincola, as these bacteria may be pathogenic. In one embodiment, the bacteria are from the sub-order micrococcuineae (micrococcuineae), including but not limited to: GRAS bacterial species Arthrobacter (Arthrobacter aritilis), Arthrobacter bailii (Arthrobacter berghei), Arthrobacter globiformis (Arthrobacter globiformis), Arthrobacter nicotianus (Arthrobacter nicotinoide), Cork.rhizophilus (Kocuria rhizophila), Kocuria variabilis (Kocuria variegans), Micrococcus luteus (Micrococcus luteus), Micrococcus luteus (Micrococcus lylae), Microbacterium guenense, Brevibacterium aurantii (Brevibacterium aurantiacaum), Brevibacterium casei (Brevibacterium casei), Brevibacterium linum linns (Brevibacterium linns), Brevibacterium foodbacterium parvum (Brevibacterium luteum), and Brevibacterium fermentum (Brevibacterium lactofermentum). In further embodiments, the bacteria are from the genus Staphylococcus (Staphylococcus), including but not limited to: staphylococcus epidermidis (Staphylococcus aureus), Staphylococcus albulans (s.arlettae), Staphylococcus aureus (s.auricularis), Staphylococcus capitis, Staphylococcus caprae, Staphylococcus carnosus (s.carnosus), Staphylococcus caseosa (staphyloccocus caseosa), Staphylococcus caseosa (staphyloccus caseosa), Staphylococcus chromogenes (s.chromogenes), Staphylococcus cohnii (s.cohnii), s.contrast, Staphylococcus dolphin (s.delphinii), Staphylococcus de, Staphylococcus equi (s.gastroanum), Staphylococcus felis (s.felis), Staphylococcus fuei (s.felis), Staphylococcus fuberidis (s.fleuraria), Staphylococcus gallinarum (s.fleuraria), Staphylococcus haemolyticus, Staphylococcus hominis (s.hyicus), Staphylococcus intermedium (s.intestinalis), Staphylococcus aureus (s.sphaericus), Staphylococcus aureus (s.kuchenensis), Staphylococcus aureus (s.ludwius), Staphylococcus lentus (s.lentus), Staphylococcus lentus (s.s.lentus) Staphylococcus felis (s.pettenkofer), staphylococcus fermentans (s.piscifierens), staphylococcus pseudointermedius (s.pseudointermedius), staphylococcus pseudolugdunensis (s.pseudolugdugdunensis), staphylococcus veus (s.pulvereri), s.rostra, staphylococcus saccharolyticus, staphylococcus saprophyticus (s.saprophyticus), staphylococcus schleiferi (s.schleiferi), staphylococcus squirrel (s.sciuri), staphylococcus gibbonis (s.simiae), staphylococcus simulans (s.simulans), staphylococcus stevensis (s.stepanovicii), staphylococcus succinus (s.succinus), staphylococcus parvus (s.vitulinus), staphylococcus wachii and staphylococcus (s.xylosus). In one embodiment, the bacterium is not staphylococcus aureus or staphylococcus epidermidis. In further embodiments, the bacteria are from the genus Streptococcus (Streptococcus), including but not limited to: streptococcus oligosaccharus (Streptococcus alimentarius), Streptococcus neighbored (Streptococcus adjacens), Streptococcus agalactiae (Streptococcus agalactiae), Streptococcus agalactiae (Streptococcus alactolicus) (Streptococcus agalactiae), Streptococcus iniae (Streptococcus bovis), Streptococcus kawachii (Streptococcus cactus), Streptococcus canis (Streptococcus lactiae), Streptococcus lactiae (Streptococcus cremoris), Streptococcus (Streptococcus caprae) (Streptococcus typus), Streptococcus typus (Streptococcus typus), Streptococcus constellatus (Streptococcus constellatus), Streptococcus constellatus (Streptococcus lactis), Streptococcus constellae (Streptococcus lactis), Streptococcus lactis (Streptococcus lactis), Streptococcus constellae (Streptococcus lactis) Streptococcus dysgalactiae (Streptococcus dysgalactiae), Streptococcus dysgalactiae (Streptococcus dysgalactiae subsp. Dysgalaciae), Streptococcus dysgalactiae (Streptococcus dysgalactiae subsp. Equisimilis), Streptococcus digestive tract (Streptococcus entosus), Streptococcus equi (Streptococcus equi. Equis), Streptococcus equi (Streptococcus equi. sp. Ruminorum), Streptococcus zooepidemicus (Streptococcus equi. subsp. Zuo), Streptococcus equi (Streptococcus equi. Streptococcus sp., Streptococcus faecalis), Streptococcus faecalis (Streptococcus faecalis), Streptococcus gallisepticum (Streptococcus pyogenes galli. galli), Streptococcus pyogenes gallisepticum (Streptococcus pyogenes, Streptococcus gallisepticum, Streptococcus pyogenes, Streptococcus Streptococcus suis (Streptococcus hyopneumoniae), Streptococcus icotinalis, Streptococcus infantis (Streptococcus infantis), Streptococcus iniae (Streptococcus infantis), Streptococcus infantis (Streptococcus infantis subsp.coli), Streptococcus iniae (Streptococcus iniae), Streptococcus intermedius (Streptococcus intermedius), Streptococcus enterobacter (Streptococcus intestinalis), Streptococcus lactis (Streptococcus mutans), Streptococcus lactis (Streptococcus lactis ), Streptococcus lactis (Streptococcus lactis), Streptococcus lactis (Streptococcus lactis) Streptococcus oligoermentatus (Streptococcus oligoermectins), Streptococcus oralis (Streptococcus oralis), Streptococcus parvus (Streptococcus paradoxus), Streptococcus paramyxis (Streptococcus paramastusis), Streptococcus parvus (Streptococcus parauberis), Streptococcus parvus (Streptococcus parvus), Streptococcus pasteuris (Streptococcus pasteuris), Streptococcus mutans (Streptococcus mutans), Streptococcus multiorhii (Streptococcus mutans), Streptococcus mutans, Streptococcus pneumoniae (Streptococcus mutans), Streptococcus mutans (Streptococcus mutans Streptococcus pneumoniae, Streptococcus mutans (Streptococcus mutans), Streptococcus mutans (Streptococcus mutans Streptococcus pneumoniae, Streptococcus mutans Streptococcus salivarius (Streptococcus salivarius), Streptococcus mutans (Streptococcus mutans) Streptococcus salivarius thermophilus (Streptococcus salivarius subsp. Thermophilus), Streptococcus sanguinis (Streptococcus sanguinis), Streptococcus mutans shiloi, Streptococcus china (Streptococcus sinensis), Streptococcus sobrinus (Streptococcus sobrinus), Streptococcus suis (Streptococcus suis), Streptococcus thermophilus (Streptococcus thermophilus), Streptococcus johnsonii (Streptococcus mutans), Streptococcus zurich (Streptococcus tiu-gurinus), Streptococcus mutans, Streptococcus uberis (Streptococcus mutans), Streptococcus curaini, Streptococcus uberis (Streptococcus mutans), Streptococcus vestibuli (Streptococcus vestibulitis), Streptococcus vestibuli (Streptococcus vestibulus), Streptococcus vestibuli (Streptococcus mutans) and Streptococcus mutans. In further embodiments, the bacteria are from the genus Lactobacillus (Lactobacillus), including but not limited to: lactobacillus gasseri (Lactobacillus garvieae), Lactococcus lactis (Lactobacillus lactis), Lactococcus lactis subsp (Lactobacillus lactis), Lactococcus lactis subsp (Lactobacillus lactis subsp. cremoris), Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. lactis, Lactococcus piscicola (Lactobacillus piscicola), Lactococcus plantarum (Lactobacillus plantarum), Lactococcus raffinosus (Lactobacillus raffinosus), Lactobacillus acidi (Lactobacillus plantarum), Lactobacillus acidi (Lactobacillus acidi), Lactobacillus acidophilus (Lactobacillus acidophilus), Lactobacillus sushi (Lactobacillus), Lactobacillus bifidus (Lactobacillus bifidus), Lactobacillus plantarum (Lactobacillus acidophilus), Lactobacillus acidophilus (Lactobacillus acidophilus), Lactobacillus bifidus (Lactobacillus acidophilus), Lactobacillus acidophilus (Lactobacillus acidophilus), Lactobacillus acidophilus, Lactobacillus (Lactobacillus) Lactobacillus bifermentatus (Lactobacillus bifermentatus), Lactobacillus brevis (Lactobacillus brevis), Lactobacillus buchneri (Lactobacillus buchneri), Lactobacillus bulgaricus (Lactobacillus bulgaricus), Lactobacillus kannieri (Lactobacillus carnis), Lactobacillus casei (Lactobacillus casei), Lactobacillus nonfermented lactococcus lactis (Lactobacillus casei), Lactobacillus casei subspecies (Lactobacillus subspecies), Lactobacillus casei (Lactobacillus casei), Lactobacillus pseudosubspecies casei (Lactobacillus casei subspecies), Lactobacillus rhamnosus subspecies (Lactobacillus subspecies), Lactobacillus casei (Lactobacillus casei), Lactobacillus casei, Lactobacillus rhamnosus (Lactobacillus casei), Lactobacillus tenacious subspecies (Lactobacillus casei), Lactobacillus casei, Lactobacillus strain, Lactobacillus strain, strain, Lactobacillus curvatus subsp (Lactobacillus curvatus subsp. curvatus), Lactobacillus curvatus melibiosus subsp (Lactobacillus curvatus subsp. melibiosus), Lactobacillus delbrueckii subsp. delbrueckii, Lactobacillus delbrueckii subsp. lactis (Lactobacillus delbrueckii subsp. lactis), Lactobacillus buchneri (Lactobacillus delbrueckii), Lactobacillus coli (Lactobacillus farinosus), Lactobacillus plantarum (Lactobacillus), Lactobacillus glusulcus, Lactobacillus plantarum (Lactobacillus forloop), Lactobacillus casei (Lactobacillus), Lactobacillus casei (Lactobacillus plantarum), Lactobacillus casei (Lactobacillus casei), Lactobacillus casei (Lactobacillus), Lactobacillus casei (Lactobacillus casei), Lactobacillus casei (Lactobacillus, Lactobacillus casei), Lactobacillus casei (Lactobacillus), Lactobacillus casei (Lactobacillus), Lactobacillus casei, Lactobacillus), Lactobacillus casei (Lactobacillus), Lactobacillus casei (Lactobacillus), Lactobacillus casei (Lactobacillus), Lactobacillus, Lactobacillus inert (Lactobacillus iners), Lactobacillus intestinalis (Lactobacillus intestinalis), Lactobacillus jensenii (Lactobacillus jensenii), Lactobacillus johnsonii (Lactobacillus johnsonii), Lactobacillus kawakii (Lactobacillus kandelii), Lactobacillus kei (Lactobacillus kefir), Lactobacillus kefiri (Lactobacillus kefir), Lactobacillus malus (Lactobacillus kefiri), Lactobacillus delbrueckii (Lactobacillus kefir), Lactobacillus plantarum (Lactobacillus lactis), Lactobacillus delbrueckii (Lactobacillus leichmannii), Lactobacillus linus (Lactobacillus crispnei), Lactobacillus casei (Lactobacillus casei), Lactobacillus casei (Lactobacillus plantarum), Lactobacillus casei (Lactobacillus), Lactobacillus plantarum (Lactobacillus), Lactobacillus casei (Lactobacillus), Lactobacillus casei (Lactobacillus), Lactobacillus casei (Lactobacillus), Lactobacillus casei (Lactobacillus), Lactobacillus case, Lactobacillus paracasei (Lactobacillus paracasei), Lactobacillus paracasei subsp (Lactobacillus paracasei subsp. paracasei), Lactobacillus paracasei (Lactobacillus paracasei subsp. tolerans), Lactobacillus paracasei (Lactobacillus paracasei), Lactobacillus plantarum (Lactobacillus paracasei), Lactobacillus pentosus (Lactobacillus pentosus, Lactobacillus pseudofood (Lactobacillus), Lactobacillus plantarum (Lactobacillus paracasei), Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus paracasei (Lactobacillus paracasei), Lactobacillus plantarum strain (Lactobacillus paracasei), Lactobacillus plantarum (Lactobacillus plantarum strain, Lactobacillus strain (Lactobacillus), Lactobacillus plantarum strain (Lactobacillus), Lactobacillus strain (Lactobacillus plantarum strain), Lactobacillus strain (Lactobacillus), Lactobacillus plantarum strain (Lactobacillus), Lactobacillus strain (Lactobacillus strain 5a), Lactobacillus strain (Lactobacillus plantarum, Lactobacillus), Lactobacillus plantarum strain (Lactobacillus), Lactobacillus sakum) Lactobacillus salivarius, Lactobacillus salivaius, Lactobacillus salivarius subsp, Lactobacillus sanfranciscensis (Lactobacillus sanfranciscensis), Lactobacillus saxiella (Lactobacillus sharpeensis), Lactobacillus thuringiensis (Lactobacillus suebicus), Lactobacillus plantarum (Lactobacillus trichoderma), Lactobacillus gingivalis (Lactobacillus saxiella), Lactobacillus plantarum (Lactobacillus sanfranciscensis), Lactobacillus plantarum (Lactobacillus vacciosus), Lactobacillus vaginalis (Lactobacillus vagaricus), Lactobacillus viridis (Lactobacillus virescens), Lactobacillus bovis (Lactobacillus sanscens), Lactobacillus plantarum (Lactobacillus sancticus), Lactobacillus plantarum (Lactobacillus sanctirium), Lactobacillus plantarum (Lactobacillus sancticus), Lactobacillus plantarum (Lactobacillus sancticus), Lactobacillus sancticus (Lactobacillus sancticus), Lactobacillus sancticus). In further embodiments, the bacteria are from the genus Lactococcus (Lactococcus), including but not limited to: lactococcus Schleifer, Lactococcus chunkiensis, Lactococcus freundii (Lactococcus fucius), Lactococcus garvieae, Lactococcus lactis subsp.

In yet another embodiment, the present disclosure provides a probiotic composition for topical delivery comprising the ns commensal skin bacteria of the present disclosure. In one embodiment, the CoNS bacterium comprises a bacterium that produces an AIP polypeptide and/or a compound of formula I. In a further embodiment, the topical composition comprises only a single microbial species that produces an AIP polypeptide or a compound of formula I. In yet further embodiments, the symbiotic skin bacteria of the present disclosure comprises a microorganism selected from the group consisting of: staphylococcus epidermidis A11, Staphylococcus hominis A9, Staphylococcus hominis C4, Staphylococcus hominis C5 and Staphylococcus Wauteri G2. In yet further embodiments, the topical probiotic composition of the present disclosure may comprise or consist of symbiotic skin bacteria selected from: staphylococcus epidermidis a11, staphylococcus hominis a9, staphylococcus hominis C4, staphylococcus hominis C5, staphylococcus woolli G2 and any combination thereof.

Commensal bacteria of the present disclosure can be isolated from human skin and identified using the methods described herein. For example, the present disclosure provides a method of obtaining, identifying, and culturing the commensal bacteria described herein by swabbing a human skin surface with, for example, a foam head swab. The swab was placed in tryptic soy broth. The broth was diluted onto mannitol agar plates (MSA) supplemented with 3% egg yolk. Pink colonies representing coagulase-negative staphylococcus (CoNS) strains without halos were collected and grown in Tryptic Soy Broth (TSB), and then 25% volume of sterile-filtered supernatant was added to Staphylococcus aureus agr type I YFP reporter strains grown in fresh TSB (for measuring inhibition of Staphylococcus aureus agr activity after 24h incubation). The agr activity of the staphylococcus aureus reporter strain was measured using a fluorimeter. Strains with strong inhibitory effect on agr activity of staphylococcus aureus were further characterized by gDNA isolation and sequencing. gDNA is isolated using any number of commercially available kits (e.g., DNeasy UltraClean microbiological Kit, Qiagen). Two cycles of sequencing of gDNA can be performed using various sequence platforms (e.g., MiSeq; Illumin Inc., San Diego, Calif.), which can generate paired-end reads of 2x250 bp. The linker is removed using cutdata (see, e.g., world-wide-web, cutdata. Low quality sequences can be removed using Trim gallore (see, e.g., world-wide-web, biolinformatics, babraham, ac, uk/projects/Trim _ bulk /) with default parameters. Sequences mapped to the human genome were removed from the linker-depleted (quality-refined) dataset using the Bowtie2 program (version 2.28) (I) with parameters (-D20-R3-N1-L20-very-sensitive-local) and the human reference genome hg 19. The filtered reads were assembled from scratch using SPAdes (version 3.8.0) with k-mers in the length range 33-127. The genome was annotated with default parameters by rapid annotation of the microbial genome using subsystem technology (RASY). The amino acid sequence (encoding DNA sequence) from the annotated CDS was aligned with the bacterial agr protein obtained from the Uniprot database. The agr genes from the assembled genome were identified according to the following three criteria: (i) sequence identity > 60%; (ii) e value < e 100; and (iii) agr locus structure, an operon of four genes, agrBDCA. Microorganisms containing sequences having at least 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% identity to the sequences of SEQ ID NO 1 or 3 are useful in the methods and compositions of the present disclosure.

As used herein, the term "probiotic composition" or "topical probiotic composition" or "probiotic skin composition" includes such compositions: the compositions include probiotic commensal skin bacteria, probiotic commensal skin bacteria fermentation extracts, attenuated or engineered microorganisms that express AIP polypeptides, and an agent that (i) inhibits protease activity or (ii) promotes protease activity, and a drug carrier that maintains viability of the commensal skin bacteria.

As used herein, the term "topical" may include administration to the skin from the outside as well as shallow injections (e.g., intradermal and intralesional) such that the topical probiotic composition is in direct contact with the skin.

As used herein, the term "fermented extract" refers to the product of fermenting probiotic commensal skin bacteria in culture under suitable fermentation conditions, for example, culture of staphylococcus aureus can produce PSM α 3 for increasing skin barrier permeability the extract of staphylococcus aureus contains PSM α 3, which can be applied to the skin to improve permeability, induce skin remodeling, or promote skin barrier permeability for drug delivery.

As used herein, the term "probiotic commensal skin bacteria" includes microorganisms of the skin microflora.a probiotic commensal skin bacteria may include a composition of bacteria that promote protease activity ("protease-promoting probiotic commensal skin bacteria"). a protease-promoting probiotic commensal skin bacteria is typically skin bacteria that produce a phenolic soluble component α (PSM α.) a protease-promoting probiotic commensal skin bacteria composition (or a fermentation extract thereof) may be used, for example, to promote skin remodeling, wound repair, aging, sunburn, pigment abnormalities, and scarring.

In further embodiments, the probiotic commensal skin bacteria may include a composition of bacteria that inhibit protease activity ("protease-inhibiting probiotic commensal skin bacteria"). Probiotic symbiotic skin bacteria compositions that inhibit proteases are useful for the treatment of diseases such as rosacea, atopic dermatitis, and netherton syndrome. In one embodiment, the probiotic symbiotic skin bacteria that inhibit proteases comprise one or more bacteria that inhibit serine protease activity of other skin bacteria and/or inhibit serine protease activity of the skin. For example, the protease-inhibiting probiotic commensal skin bacteria may comprise coagulase-negative staphylococcal species. In one embodiment, the coagulase-negative strain is selected from the group consisting of: staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus saccharolyticus, Staphylococcus wowensis, Staphylococcus pasteuri, Staphylococcus haemolyticus, Staphylococcus delbrueckii, Staphylococcus hominis, Staphylococcus aureus jettentis, Staphylococcus petasii and Staphylococcus lugdunensis. In one embodiment, the protease-inhibiting commensal skin bacterium is selected from the group consisting of a staphylococcus epidermidis strain, a staphylococcus hominis strain, a staphylococcus wovensis strain, and any combination thereof. In a particular embodiment, the staphylococcus epidermidis strain is staphylococcus epidermidis 14990 and/or staphylococcus epidermidis a 11. In further embodiments, the human staphylococcus strain is human staphylococcus C4, human staphylococcus C5, and/or human staphylococcus a 9. In another specific embodiment, the staphylococcus wadwinii strain is staphylococcus wadwinii G2. In one embodiment, the CoNS bacterium comprises a bacterium that produces an AIP polypeptide and/or a compound of formula I. In a further embodiment, the topical composition comprises only a single species of microorganism that produces an AIP polypeptide or a compound of formula I. In yet further embodiments, the symbiotic skin bacteria of the present disclosure comprises a microorganism selected from the group consisting of: staphylococcus epidermidis A11, Staphylococcus hominis A9, Staphylococcus hominis C4, Staphylococcus hominis C5 and Staphylococcus Wauteri G2. In yet further embodiments, the topical probiotic composition of the present disclosure may comprise or consist of symbiotic skin bacteria selected from: staphylococcus epidermidis a11, staphylococcus hominis a9, staphylococcus hominis C4, staphylococcus hominis C5, staphylococcus woolli G2 and any combination of the foregoing.

The term "contacting" refers to exposing the skin to a topical probiotic composition such that the probiotic skin composition may modulate protease activity (e.g., serine protease activity) on the skin.

The term "inhibiting" or "inhibiting effective amount" refers to an amount of a probiotic skin composition consisting of one or more probiotic microorganisms and/or fermentation medium or extract and/or fermentation by-products and/or synthetic molecules sufficient to inhibit protease activity (e.g. serine protease activity), e.g. on the skin or in skin culture. The term "inhibiting" also includes preventing or ameliorating signs or symptoms of a disorder (e.g., rash, pain, etc.).

As used herein, for treating a subject having a disease or disorder, the term "therapeutically effective amount" refers to an amount of the probiotic skin composition or extract thereof sufficient to ameliorate the signs or symptoms of the disease or disorder. For example, by measuring the frequency of the severity of skin pain, the therapeutically effective amount can be measured as an amount sufficient to alleviate symptoms of dermatitis or rash in a subject. Typically, the subject is treated in an amount that reduces the symptoms of the disease or disorder by at least 50%, 90%, or 100%. Generally, the optimal dosage will depend on the condition and various factors (e.g., the weight of the subject, the type of bacteria, the sex of the subject, and the degree of symptoms). However, one skilled in the art can readily determine the appropriate dosage.

As used herein, the terms "purified" and "substantially purified" refer to cultures or co-cultures of microorganisms or biological agents (e.g., fermentation media and extracts, fractionated fermentation media, fermentation byproducts, AIP peptides, polypeptides, genes, polynucleotides, compounds of formula I, etc.) that are substantially free of other components found in the cell or natural environment naturally associated with the agent produced in vivo. In some embodiments, the co-cultured probiotic may comprise a plurality of commensal skin bacteria.

The present disclosure provides a whole cell preparation comprising a substantially homogeneous preparation of staphylococcus epidermidis, staphylococcus hominis and/or staphylococcus waderi. Such formulations may be used to prepare compositions for the treatment of inflammation and microbial infections. Whole cell preparations may comprise staphylococcus epidermidis, staphylococcus hominis and/or staphylococcus woolli, or may comprise a non-pathogenic (e.g., attenuated microorganism) carrier as described below. The present disclosure also provides a fraction derived from such whole cells (fraction) comprising an agent that reduces protease activity in skin due to staphylococcus aureus activity.

The ability of a first bacterial composition to inhibit the protease activity of a second bacterial composition can be determined by measuring the protease activity of the second bacterial composition before and after contacting the second composition with the first composition. Contacting of the organism with the topical probiotic composition of the present disclosure may occur in vitro, for example, by adding the topical probiotic composition to a bacterial culture to test the bacteria for protease inhibitory activity. Alternatively, the contacting may occur in vivo, for example, by contacting the topical probiotic composition with a subject suffering from a skin disease or disorder.

Probiotic symbiotic skin bacteria formulations may be prepared in a variety of ways. Any of a variety of methods known in the art may be used to administer the topical probiotic composition to a subject. For example, the probiotic skin compositions or extracts or synthetic formulations of the present disclosure may be formulated for topical administration (e.g., as a lotion, cream, spray, gel, or ointment). Such topical formulations are useful for treating or inhibiting the presence of microorganisms, fungi, viruses or infections or inflammations on the skin. Examples of formulations include topical lotions, creams, soaps, wiping sheets, and the like.

In another embodiment, a topical probiotic composition is provided comprising a plurality of probiotic commensal skin bacteria, when used to treat dermatitis or other skin disease or condition associated with increased protease (e.g., serine protease) activity, the composition comprising one or more bacteria that inhibit protease activity on the skin, in which case the probiotic commensal skin bacteria are coagulase-negative staphylococcal species.

In further embodiments, the topical probiotic composition comprises a probiotic symbiotic skin bacteria fermented extract that promotes protease activity on the skin. In various aspects, the bacteria from which the extract is produced comprise staphylococcus aureus.

In yet further embodiments, topical probiotic compositions are provided that consist essentially of a staphylococcus aureus fermentation extract (alone, or in combination with staphylococcus aureus). According to another aspect, the above topical probiotic composition may be formulated as a lotion, a shake, a cream, an ointment, a gel, a foam, a powder, a solid, a paste or a tincture.

In further embodiments, the topical probiotic composition comprises a probiotic symbiotic skin bacteria fermented extract. In various aspects, the extract-producing bacteria comprise a coagulase-negative staphylococcus species. In one embodiment, the staphylococcus species is selected from the group consisting of: a staphylococcus epidermidis strain, a staphylococcus hominis strain, a staphylococcus wovensis strain, and any combination thereof, which produces AIP that inhibits protease production in the agr quorum sensing system and/or in the microbiota of the skin. In one embodiment, the AIP comprises the consensus sequence of SEQ ID NO 10, or a sequence having at least 98% identity to

SEQ ID NO

4, 11, 12, 13, 14, 15, 16 or 17 having agr population modulating activity, and/or a compound of formula I, IA or IB.

In yet further embodiments, topical probiotic compositions are provided that consist essentially of a coagulase-negative staphylococcus species fermented extract or a staphylococcus epidermidis fermented extract (alone, or in combination with a coagulase-negative staphylococcus species or staphylococcus epidermidis). In additional embodiments, the composition comprises one or more of the deposited microbial strains described herein (e.g., staphylococcus epidermidis a11, staphylococcus hominis a9, staphylococcus hominis C5, and/or staphylococcus wavorans G2).

According to another embodiment, the above topical probiotic composition may be formulated as a lotion, a shake, a cream, an ointment, a gel, a foam, a powder, a solid, a paste or a tincture.

In a further embodiment, a fermentation extract is provided, which may be obtained by fermenting under fermentation conditions a bacterium selected from the group consisting of: a staphylococcus epidermidis strain, a staphylococcus hominis strain, a staphylococcus wovensis strain, and any combination thereof. In various aspects, such fermented extracts can be used to inhibit serine protease activity on skin. In further embodiments, the fermented extract is obtained from any one or more of the deposited microbial strains described herein (e.g., staphylococcus epidermidis a11, staphylococcus hominis a9, staphylococcus hominis C5, and/or staphylococcus wavorans G2). According to another embodiment, the fermented extract may be formulated as a lotion, a shake, a cream, an ointment, a gel, a foam, a powder, a solid, a paste or a tincture.

In further embodiments, a bandage or dressing is provided comprising the above-described topical probiotic composition, the above-described probiotic symbiotic skin bacteria fermented extract, the above-described probiotic symbiotic skin bacteria, and any combination thereof. In various aspects, a bandage or dressing is provided whose main ingredients include a substrate and probiotic commensal skin bacteria that inhibit protease activity on the skin. In various aspects, a bandage or dressing is provided whose main ingredients include a substrate and a probiotic symbiotic skin bacteria fermentation extract that inhibits protease activity on the skin.

In further embodiments, a bandage or dressing is provided comprising the above-described topical probiotic composition, the above-described probiotic symbiotic skin bacteria fermented extract, the above-described probiotic symbiotic skin bacteria, and any combination thereof. In various aspects, a bandage or dressing is provided whose main ingredients include a substrate and probiotic commensal skin bacteria that promote protease activity on the skin. In various aspects, a bandage or dressing is provided whose main ingredients include a substrate and a probiotic symbiotic skin bacteria fermentation extract that promotes protease activity on the skin.

The present disclosure also provides a method for treating a skin disease or condition associated with a protease (e.g., serine protease activity). Examples of such diseases or disorders include Netherton syndrome, atopic dermatitis, contact dermatitis, eczema, psoriasis, acne, epidermal hyperkeratosis, acanthosis, epidermal inflammation, dermal inflammation and pruritus. In one embodiment, the presence of a disease or disorder is first determined by measuring protease activity of a sample (e.g., a sample of skin or a bacterial culture from skin) from a subject suspected of having the disease or disorder. If the sample exhibits a higher than normal protease activity (e.g., serine protease activity), the subject is treated with the protease-inhibiting symbiotic bacterial preparation by contacting the subject's skin with the preparation. In further embodiments, a culture from a subject with high protease activity and comprising bacteria is contacted with the preparation in vitro to determine the sensitivity of the culture to the preparation and its effect on protease inhibition.

The protease-inhibiting symbiotic bacterial preparation or fermentation extract may be combined with one or more known serine protease inhibitors. There are a number of commercially and clinically relevant serine protease inhibitors that can be used in the methods and compositions of the present disclosure. For example, serine protease inhibitors, such as those disclosed in, for example, U.S. patent No. 5,786,328, U.S. patent No. 5,770,568, or U.S. patent No. 5,464,820, the disclosures of which are incorporated herein by reference. Exemplary serine protease inhibitors include antibodies that bind to and inhibit a serine protease polypeptide or functional fragment thereof, enzymes that degrade a serine protease polypeptide into inactive peptides, substrate analogs, and the like. Serine protease expression inhibitors include, for example, antisense molecules, ribozymes, and small molecule agents (e.g., vitamin D antagonists) that reduce transcription or translation of a serine protease polynucleotide (e.g., DNA or RNA). One embodiment of the present disclosure relates to substrate analogs of tissue kallikrein. These substrate analogs comprise peptides having an amino acid sequence corresponding to positions 388 to 390 of tissue kallikrein. Peptides can be prepared synthetically by recombinant engineering techniques, e.g., by cloning and expressing nucleic acid sequences, or purified from natural sources such as bacteria, fungi, or cell extracts. The structural, chemical, physicochemical, nomenclature, and analytical aspects of Amino Acids are compiled in "Amino Acids Chemistry" (J.P.Greenstein and M.Winitiz, John Wiley&Sons, New York, n.y., rev 1961,1984), which is expressly incorporated herein by reference. Peptides include modified and/or unmodified amino acids, including naturally occurring amino acids, non-naturally occurring (non-coding) amino acids, synthetically prepared amino acids, and combinations thereof. Naturally occurring amino acids include glycine (Gly), amino acids having alkyl side chains such as alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile) and proline (Pro), aromatic amino acids phenylalanine (Phe), tyrosine (Tyr) and tryptophan (Trp), amino acid alcohol serine (Ser)) And threonine (Thr), the acidic amino acids aspartic acid (Asp) and glutamic acid (Glu), the amides of Asp and Glu, asparagine (Asn) and glutamine (gin), the sulfur-containing amino acids cysteine (Cys) and methionine (Met), and the basic amino acids histidine (His), lysine (Lys) and arginine (Arg). Non-naturally occurring amino acids include, for example, ornithine (Orn), norleucine (Nle), citrulline (citrulline) (Cit), homocitrulline (hCit), desmosine (Des), and isodesmosine (Ide). Modified amino acids include naturally and non-naturally occurring amino acids as well as synthetically produced derivatives and analogs of the amino acids. Such amino acid forms have been chemically modified, for example, by halogenating one or more active sites with chlorine (Cl), bromine (Br), fluorine (F), or iodine (I); with carbon-containing groups, e.g. methyl (Me), ethyl (Et), butyl (Bu), amino (NH)₂Or NH₃) Examples include amino acid hydroxamates and decarboxylases, dansyl amino acids, polyamino acids and amino acid derivatives specific examples include Gamma Amino Butyric Acid (GABA), hydroxyproline (Hyp), aminoadipic acid (Aad) which may be modified at position 2 or 3, O-aminobutyric acid (Aab or Abu), selenocysteine (SeCys2), tert-butylglycine (Bug or Buter-BuGly), N-carbamoylamino acid, amino methyl ester, aminopropionic acid (or β -alanine; 13-Ala), adamantylglycine (Adg), aminocaproic acid (Acp), N-ethylaspartamide (Et-Asn), allo-hydroxyisoleucine (aL), allo-phenylalanine (HyaIle), phenylalanine (Phe-alanine), aminoalanine (Phe-2-alanine) (Ile-2-Ile-D-Phe), aminoalanine (Phe-2-D-Ile), aminoalanine (Phe-D-2-D-Ile), aminoalanine (Phe-D-2-Ile), aminoalanine (Phe-DNon-coding amino acids include, for example, phenylglycine (Ph-Gly), cyclohexylalanine (Cha), cyclohexylglycine (Chg), and 4-aminophenylalanine [ Phe (4 NH)₂) Or Aph]. The modified amino acid may also be of the chemical structure: the chemical structure is not an amino acid at all, but is actually classified into another chemical form, such as an alkylamine, sugar, nucleic acid, lipid, fatty acid, or other acid. Any modified or unmodified amino acid comprising a peptide may be in the D-or L-conformation, or comprise one, two or more tautomers or synthons.

A pharmaceutical composition comprising a probiotic skin composition disclosed herein comprising commensal bacteria (e.g., staphylococcus epidermidis a11, staphylococcus hominis a9, staphylococcus hominis C4, staphylococcus hominis C5 and/or staphylococcus wavorans G2), engineered forms thereof (e.g., attenuated or genetically modified forms), or attenuated microorganisms containing AIP peptide coding sequences, may be formulated in any dosage form suitable for topical administration to produce a local or systemic effect, including emulsions, solutions, suspensions, creams, gels, hydrogels, ointments, dusting powders, dressings, elixirs, lotions, suspensions, tinctures, pastes, foams, films, aerosols, rinses, sprays, suppositories, bandages, skin patches. Topical formulations comprising the probiotics disclosed herein may also comprise liposomes, micelles, microspheres, nanosystems and mixtures thereof.

In one embodiment, a bandage or dressing is provided comprising a probiotic skin composition disclosed herein, which comprises a commensal bacterium (e.g., staphylococcus epidermidis a11, staphylococcus hominis a9, staphylococcus hominis C4, staphylococcus hominis C5 and/or staphylococcus woolli G2), an engineered form thereof (e.g., an attenuated or genetically modified form), or an attenuated microorganism containing an AIP peptide coding sequence as described herein. In various aspects, a bandage or dressing is provided whose major components include a substrate and a probiotic skin composition comprising a commensal bacterium (e.g., staphylococcus epidermidis a11, staphylococcus hominis a9, staphylococcus hominis C4, staphylococcus hominis C5 and/or staphylococcus woolli G2), an engineered form thereof (e.g., an attenuated or genetically modified form), or an attenuated microorganism containing the AIP peptide coding sequence described above. In various embodiments, a bandage or dressing is provided whose major ingredients include a substrate and a probiotic symbiotic skin bacteria or extract. In various aspects, a bandage or dressing is provided having as a primary component a substrate and a probiotic symbiotic skin bacteria fermented extract. In various aspects, a bandage or dressing is provided having a base comprising a matrix and glycerin. In one embodiment, the bandage or dressing is applied to the site of skin damage or injury. In further embodiments, a bandage or dressing is applied to the site of infection.

"pharmaceutically acceptable carrier" is intended to include solvents, dispersion media, coatings, antimicrobial and antifungal agents (as long as they are not harmful to the probiotic commensal bacteria, if desired), isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the pharmaceutical composition, its use in the therapeutic compositions and methods of treatment is contemplated. Supplementary active compounds may also be incorporated into the compositions.

Pharmaceutically acceptable carriers and excipients suitable for use in the topical formulations disclosed herein include, but are not limited to, aqueous vehicles, water-miscible vehicles, non-aqueous vehicles, stabilizers, solubility enhancers, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, wetting or emulsifying agents, complexing agents, sequestering or chelating agents, penetration enhancers, cryoprotectants, lyoprotectants, thickening agents, and inert gases.

The pharmaceutical composition comprising the probiotic bacteria may be formulated in the form of ointments, creams, sprays and gels. Suitable ointment vehicles include oily or hydrocarbon vehicles including, for example, lard, benzoic lard, olive oil, cottonseed oil and other oils, white petrolatum; emulsifying or absorbing vehicles such as hydrophilic petrolatum, hydroxy glycerol tristearate sulfate, glycerin, and anhydrous lanolin; water-removable vehicles, such as hydrophilic ointments; water soluble ointment vehicles including polyethylene glycols of varying molecular weights; emulsion vehicles, water-in-oil (W/O) emulsions or oil-in-water (O/W) emulsions, including cetyl alcohol, glyceryl monostearate, lanolin and stearic acid [ see remington: pharmaceutical technology and Practice (Remington: The Science and Practice of Pharmacy) ]. These excipients are emollient but generally require the addition of antioxidants and preservatives.

Suitable cream bases may be oil-in-water or water-in-oil. The cream vehicle may be water-washable and comprises an oil phase, an emulsifier, and an aqueous phase. The oil phase, also referred to as the "internal" phase, typically comprises petrolatum and a fatty alcohol, such as cetyl or stearyl alcohol. The aqueous phase typically, although not necessarily, exceeds the oil phase in volume and typically contains a humectant. The emulsifier in the cream formulation may be a nonionic, anionic, cationic or amphoteric surfactant.

Gels are semi-solid suspension type systems. A single-phase gel comprises a material that is substantially homogeneous throughout a liquid carrier. Suitable gelling agents include cross-linked acrylic polymers, such as carbomers, carboxypolyalkylene (Carbopol), Carbopol^RTM(ii) a Hydrophilic polymers such as polyethylene oxide, polyoxyethylene-polyoxypropylene copolymer and polyvinyl alcohol; cellulose polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose phthalate and methyl cellulose; gums, such as tragacanth and xanthan gum; sodium alginate; and gelatin. To prepare a homogeneous gel, a dispersing agent such as an alcohol or glycerin may be added, or a gelling agent may be dispersed by grinding, mechanical mixing, and/or stirring.

In additional embodiments, pharmaceutical compositions comprising a compound of formula I and/or symbiotic probiotics, derivatives or analogs thereof disclosed herein can be formulated alone or in combination with one or more additional therapeutic agents including, but not limited to, chemotherapeutic agents, antibiotics (so long as they do not destroy the benefits of the probiotics), antifungal agents, antipruritic agents, analgesic agents, protease inhibitors, and/or antiviral agents.

As used herein, topical administration includes dermal (intradermal), conjunctival, intracorneal, intraocular, ocular, otic, transdermal, nasal, vaginal, urethral, respiratory, and rectal administration. Such topical formulations are useful for treating or inhibiting cancer of the eye, skin and mucous membranes (e.g., oral, vaginal, rectal). Examples of formulations on the market include topical lotions, creams, soaps, wiping sheets, etc.

The solution or suspension used in the pressurized container, pump, spray, atomizer or nebulizer may be formulated to contain ethanol, dilute alcohol or a suitable substitute for dispersing, solubilizing or extending the release of the active ingredients disclosed herein, a propellant as a solvent; and/or surfactants such as sorbitan trioleate, oleic acid or oligomeric lactic acid.

Materials that may be used to form the erodable matrix include, but are not limited to, chitin, chitosan, dextran, and pullulan; agar gum (guar gum), gum arabic, karaya gum, locust bean gum, tragacanth gum, carrageenan gum, ghatti gum (gum ghatti), guar gum, xanthan gum, and scleroglucan; starches, such as dextrin and maltodextrin; hydrocolloids, such as pectin; phospholipids, such as lecithin; alginic acid; propylene glycol alginate; gelatin; collagen; and cellulosics such as Ethyl Cellulose (EC), Methyl Ethyl Cellulose (MEC), carboxymethyl cellulose (CMC), CMEC, hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), Cellulose Acetate (CA), Cellulose Propionate (CP), Cellulose Butyrate (CB), Cellulose Acetate Butyrate (CAB), CAP, CAT, hydroxypropyl methyl cellulose (HPMC), HPMCP, HPMCAS, hydroxypropyl methyl cellulose acetate trimellitate (HPMCAT), and ethyl hydroxyethyl cellulose (EHEC); polyvinylpyrrolidone; polyvinyl alcohol; polyvinyl acetate; glycerin fatty acid ester; polyacrylamide; polyacrylic acid; copolymers of ethacrylic acid (ethacrylic acid) or methacrylic acid (EUDRAGIT, Rohm America, inc., Piscataway, n.j.); poly (2-hydroxyethyl methacrylate); a polylactide; a copolymer of L-glutamic acid and ethyl L-glutamate; degradable lactic acid-glycolic acid copolymers; poly-D- (-) -3-hydroxybutyric acid; and other acrylic acid derivatives such as homopolymers and copolymers of butyl methacrylate, methyl methacrylate, ethyl acrylate, (2-dimethylaminoethyl) methacrylate and (trimethylaminoethyl) methacrylate chloride.

In yet another embodiment, the compositions provided herein (e.g., probiotic compositions or compositions comprising peptides or compounds of formula I) can be combined with one or more steroid drugs known in the art including, but not limited to, aldosterone, beclomethasone, betamethasone, deoxycorticosterone acetate, fludrocortisone acetate, hydrocortisone (cortisol), prednisolone, prednisone, methylprednisolone, dexamethasone, and triamcinolone.

In another embodiment, the compositions provided herein (e.g., probiotic compositions or compositions comprising peptides or compounds of formula I) may be combined with one or more antifungal agents including, but not limited to, amorolfine, amphotericin B, anidulafungin, bifonazole, butenafine, butoconazole, caspofungin, ciclopirox, clotrimazole, econazole, fenticonazole, felopin, fluconazole, isoconazole, itraconazole, ketoconazole, micafungin, miconazole, naftifine, natamycin, nystatin, oxiconazole (oxyconazole), lafutizole (ravuconazole), posaconazole, rimonazol, sertaconazole, sulconazole, terbinafine, terconazole, tioconazole, and voriconazole.

Kits and articles of manufacture are also described herein for use in the therapeutic applications described herein. Such kits may include a carrier, package, or container that is compartmentalized to receive one or more containers, e.g., vials, tubes, and the like, each container comprising one of the individual elements for the methods described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The container may be formed from a variety of materials, such as glass or plastic.

For example, the container may comprise one or more of the compositions provided herein (e.g., probiotic compositions or compositions comprising peptides or compounds of formula I), optionally in combination with other agents disclosed herein. Such kits optionally comprise a composition disclosed herein, and an identifying description or label or instructions relating to the use of the composition in the methods described herein.

The following examples are provided to further illustrate the invention, but are not intended to limit the invention.

Examples

Example 1

And (3) culturing primary human keratinocytes. At 37 ℃ 5% CO₂Next, neonatal NHEK (ThermoFisher Scientific, Waltham, Mass.) was cultured in EpiLife medium (ThermoFisher Scientific) supplemented with 1 × EpiLife defined growth supplements (ThermoFisher Scientific), 60 μ M CaCl₂And 1 Xantibiotic-antifungal (PSA; 100U/ml penicillin, 100U/ml streptomycin, 250ng/ml amphotericin B; ThermoFisher scientific). For the experiment, NHEK was grown to 70% confluence and then in high calcium EpiLife medium (2mM CaCl)₂) For 48 hours, and then treated with the bacteria sterile-filtered supernatant. The use of these human-derived commercial cell products does not require informed consent. For bacterial supernatant treatment, differentiated NHEK was treated with sterile filtered bacterial supernatant (5% by volume) added to EpiLife medium. NHEK was used only in experiments between passage 3 and 5.

And (5) culturing bacteria. All bacteria were cultured at 37 ℃ in 3% tryptose soy broth (TSB; Sigma, St. Louis, Mo.) with shaking at 300 r.p.m.. Staphylococcus aureus strains Newman, USA300, 113, SANGER252 and Staphylococcus epidermidis strains ATCC12228 and ATCC1457 were grown for 24 hours to reach stationary phase, and then centrifuged (4,000r.p.m., room temperature [ RT.]10 min) and the supernatant was sterile filtered (0.22 μm) and then added to NHEK. Briefly, protease-free strains were cultured in 3% TSB containing 25. mu.g/ml lincomycin and 5. mu.g/ml erythromycin for 24 hours, followed by a further 24 hours of sub-culturing in only 3% TSB. For murine active staphylococcus aureus colonization assay, 2x10 was determined⁶Colony-forming units of bacteria were applied to 8-mm TSB agar plates, allowed to dry at room temperature for 30 minutes, and then added to the skin of the back of the rat.

Murine bacterial disc (bacterial disc) model. Female C57BJ/6L mice (8 weeks old) were used for a murine model of bacterial skin colonization. Briefly, to remove hair from the back skin, mice were shaved and nair applied for 2e3 minutes, then the hair was removed with an alcohol wipe. After 24 hours of recovery, 3x8mm TSB agar plates were applied to the skin of the rat back for 12 hours, with each plate containing TSB alone (vehicle control) or each plate containing 2e⁶Colony forming units of Staphylococcus aureus (USA 300). Tegaderm was applied on top of the agar plate to immobilize it. Mice were euthanized and 8-mm whole skin needle biopsies were collected for analysis.

In situ zymography. Sections of murine skin (10 μm thick) were washed 1 time with 1% tween-20 in water for 5 minutes. Sections were treated with 2 μ g ml of BODIPY FL total protease activity substrate (Thermo-Fisher scientific) at 37 ℃ for 4 hours in a humidification chamber to measure total protease activity. Serine protease inhibitor AEBSF (50 mM; Sigma) was also applied to the sections 30 minutes before the addition of BODIPY FL casein. Slides were rinsed 1 time in phosphate buffered saline, then a gold anti-fluorescence quenching coverslipping (gold fluorescent quenching medium) without DAPI and coverslips were applied. The fluorescence signal was measured using an Olympus BX51(Tokyo, Japan) fluorescence microscope.

And (4) protease activity determination. NHEK conditioned medium was added in 50ml to a 96 well black plate (Corning, Corning, NY) followed by 150ml of 5. mu.g ml BODIPY FL casein substrate, 2. mu.g ml elastin (elastase-like substrate; ThermoFisher Scientific) or 4. mu.g ml gelatin (MMP substrate; ThermoFisher Scientific) according to the manufacturer's instructions. In addition, 200. mu.M of the peptide Boc-Val-Pro-Arg-AMC (trypsin-like substrate; BACHEM, Bubendorf, Switzerland) was added to NHEK conditioned medium in 150. mu.l of 1 Xdigestion buffer (ThermoFisher Scientific). Relative fluorescence intensity was analyzed by a SpectraMAX Gemini EM fluorometer (ThermoFisher Scientific) at room temperature, reading every 2 hours for 24 hours. BODIPY FL casein white plate was read at ex:485nm and em:530 nm. The elastin-like and MMP substrate plates were read at ex:485nm and em:515 nm. The trypsin-like substrate plate was read at ex:354nm and em:435 nm.

And (5) real-time quantitative PCR. RNA was isolated from NHEK using Purelink RNA isolation column (ThermoFisherScientific) according to the manufacturer's instructions. RNA was quantified using a Nanodrop spectrophotometer (ThermoFisher Scientific) and 500ng of RNA was reverse transcribed using the iScript cDNA synthesis kit (Bio-Rad, Irvine, Calif.). Real-time quantitative PCR reactions were performed on a CFX96 real-time detection system (Bio-Rad) using gene-specific primers and TaqMan probes (ThermoFisher Scientific).

Immunoblotting. For Cell lysis, cold 1x radioimmunoprecipitation assay (RIPA) buffer (Sigma) containing a 1x protease inhibitor cocktail (Cell signaling technology, Danvers, MA) was applied to the NHEK followed by scraping (scraping). The cell lysate was incubated on ice for 30 minutes and then centrifuged (13,000r.p.m., 15 minutes, 4 ℃) to remove cell debris. The samples were prepared as follows: protein concentration was determined using a bicinchoninic acid (BCA) assay (Pierce, Rockford, Ill.), then 40mg of sample was added to 4X Laemmli sample buffer (Bio-Rad) containing 1% b-mercaptoethanol and heated at 95 ℃ for 7 minutes. Samples were electrophoresed on 4-20% Tris-glycine precast TGX gels (Bio-Rad), transferred to 0.22- μm polyvinylidene fluoride (PVDF) membranes (Bio-Rad) using a trans-blot turbo transfer system (Bio-Rad), blocked in a 1x odyssey blocking solution (LI-COR, Lincoln, NE) containing 0.1% tween-20 for 1 hour at room temperature, and stained with primary antibody at 4 ℃ overnight. After washing with 3x PBST (phosphate buffered saline containing 0.1% tween-20), Odyssey (LI-COR) fluorescent secondary antibody was applied to the membrane for 1 hour at room temperature on an orbital shaker. Additional 3x PBST washes were performed prior to analysis on an infrared imager (LI-COR). Primary antibodies from Santa Cruz Biotechnologies (Santa Cruz, Calif.) KLK5(H-55), KLK6(H-60), DSG-1(H-290), FLG (H-300) and a-tubulin (TU-02) were used at a dilution of 1: 100. KLK13(ab28569) and KLK14(ab128957) antibodies from Abcam (Cambridge, UK) were used at a dilution of 1:1,000.

KLK gene silencing. Cultivation Using RNAimax (ThermoFisher scientific) and OptiMEMBased on (ThermoFisher Scientific), either siRNA or siRNA scrambled (-) controls (ThermoFisher Scientific) were selected with specific KLK silencers at 15nM or 45nM and NHEK was treated for 24 hours. NHEK was cultured on high calcium medium (2 mMCaCl)₂) Medium differentiation was performed for 48 hours, followed by 24 hours treatment with sterile filtered staphylococcus aureus (Newman) supernatant before analysis of NHEK lysates and conditioned media.

And (5) carrying out statistical analysis. Both one-way and two-way anova were used for statistical analysis, with P values <0.05 being significant. Statistical analysis of the results was performed using GraphPad prism version 6.0 (GraphPad, La Jolla, CA).

Staphylococci affect the protease activity of human keratinocytes. To assess whether different bacterial strains found on human skin could induce keratinocyte protease activity, primary cultures of Normal Human Epidermal Keratinocytes (NHEK) were treated with sterile-filtered culture supernatants from four different laboratory isolates of staphylococcus aureus, including two methicillin-resistant staphylococcus aureus strains (USA300 and SANGER252) and two methicillin-sensitive staphylococcus aureus strains. Two staphylococcus epidermidis isolates (ATCC12228 and ATCC1457) were also tested. Twenty-four hours after exposure to sterile bacterial culture supernatants, keratinocyte cell culture media were analyzed for protease activity, and substrate selection for trypsin-like, elastase-like, or Matrix Metalloproteinase (MMP) activity. NHEK conditioned medium contained significantly more trypsin activity after treatment with staphylococcus aureus strain Newman and USA300 (fig. 1 a). Staphylococcus epidermidis strain ATCC12228 increased MMP and elastase activities, while staphylococcus aureus strain USA300 and SANGER252 and staphylococcus epidermidis strain ATCC1457 increased elastase activities in NHEK-conditioned medium to a lesser extent (fig. 1b and c). To confirm that the increased protease activity observed in NHEK-conditioned media was derived from NHEK, and not produced by the bacteria themselves, trypsin activity was analyzed after addition of staphylococcus aureus (Newman) supernatant to the culture wells in the presence and absence of NHEK. When the same concentration of diluted supernatant from staphylococcus aureus was added to NHEK medium alone, no enzyme activity was detected in the absence of NHEK (fig. 1 d).

Staphylococcus aureus increases epidermal serine protease activity. Because of the large increase in trypsin activity induced by certain staphylococcus aureus strains (Newman and USA300), and the potential role of this activity in diseases mediated by staphylococcus aureus, experiments were conducted on this organism to better understand how the bacteria induce protease activity in NHEK. To evaluate the kinetics of the protease response to staphylococcus aureus, keratinocytes were treated with sterile-filtered culture supernatant from staphylococcus aureus (Newman) for 0, 8, 24, and 48 hours before collecting NHEK conditioned media for protease analysis. Measurements of total protease activity in NHEK conditioned medium showed that total proteolytic activity increased with time after exposure to staphylococcus aureus supernatant (fig. 2 a). The addition of the serine protease inhibitor aprotinin confirmed that this activity was due to serine protease (fig. 2b), which is consistent with the observation of increased trypsin-like activity shown in fig. 1 a. Comparison of s.aureus USA300LAC wild-type and protease-free strains showed that both wild-type and protease-free strains increased trypsin activity in NHEK conditioned medium, but that the protease-free strain had a significantly reduced ability to induce trypsin activity compared to the wild-type strain (fig. 2 c). Taken together, these data demonstrate that staphylococcus aureus can increase endogenous NHEK serine protease activity, and that staphylococcus aureus protease and other staphylococcus aureus products contribute to the ability of the bacterium to activate keratinocytes.

To further verify the effect of staphylococcus aureus on epidermolytic activity, active staphylococcus aureus (USA300) was applied to the dorsal skin of mice. The skin at the application site was then biopsied and sectioned for analysis of total proteolytic activity by in situ zymography in the presence or absence of the serine protease inhibitor 4-benzenesulfonyl fluoride (AEBSF). The total epidermal protease activity in the epidermis was qualitatively improved after treatment with staphylococcus aureus compared to skin treated with agar plates only, and the increased activity detected by fluorescence enhancement was largely eliminated by inhibition of serine protease activity with AEBSF. Background autofluorescence at the hair follicle was observed in all sections including the no-substrate control. These observations further demonstrate that the presence of staphylococcus aureus can increase protease activity in the epidermis.

Staphylococcus aureus increases KLK expression in keratinocytes. KLK is a family of serine proteases abundant in the epidermis, with trypsin-like or chymotrypsin-like activity. To determine whether s.aureus can alter KLK mRNA expression in keratinocytes, NHEK was treated with s.aureus (Newman) supernatant for 24 hours and KLK1-15 expression was measured by real-time quantitative PCR. KLK5 was the most abundant relative mRNA, whereas KLK6, 13 and 14 consistently showed the greatest fold increase after exposure to staphylococcus aureus (fig. 3 a-e). All other KLKs analyzed showed a slight increase in mRNA expression after exposure to staphylococcus aureus, except KLK1 showed a decrease in expression. No mRNA of KLK2, 3, and 15 was detected.

Cell lysates and NHEK conditioned media were then analyzed for changes in KLK protein expression after staphylococcus aureus (Newman) supernatant treatment. Immunoblots of KLK6 and 14 showed increased expression of these KLK proteins after staphylococcus aureus supernatant treatment in cell lysates and conditioned media, whereas KLK13 was increased only in conditioned media. Expression of KLK5 was unchanged after staphylococcus aureus supernatant treatment (fig. 3 f).

KLK6, 13 and 14 contribute to increase keratinocyte serine protease activity. Since KLK6, 13 and 14 showed the greatest increase in expression in NHEK after s.aureus exposure, experiments were performed to examine whether these KLKs were associated with the observed increase in serine protease activity. Small interfering RNAs (siRNAs) are useful for selectively silencing their expression. siRNAs to KLK6 and KLK13 significantly reduced trypsin activity induced by Staphylococcus aureus, while KLK14 reduced trypsin activity to a lesser extent. Triple knockdown of KLK6, 13 and 14 also showed a significant reduction in trypsin activity relative to control siRNA, although no additive effect was observed (fig. 4 a). Interestingly, the triple knockdown of KLK6, 13 and 14 resulted in a reduction in the knockdown efficiency of KLK13 and KLK14, which could explain the lack of additive effects on trypsin activity (fig. 4 b-d).

Staphylococcus aureus promotes degradation of desmoglein-1 and FLG by inducing KLK. Desmoglein-1 (DSG-1) and FLG are both important in regulating the integrity of the epidermal skin barrier. Immunoblotting showed that exposure of NHEK to Staphylococcus aureus (Newman) supernatant promoted cleavage of full-length DSG-1(160kDa), while siRNA silencing of KLK6, 13, or 14 blocked cleavage of DSG-1 (FIG. 5 a). siRNA silencing of KLK6 and KLK13 also partially blocked s.aureus-mediated cleavage of profilaggrin (Pro-FLG) in NHEK (indicated by the band >250kDa on the immunoblot) (fig. 5 b). Densitometric analysis further demonstrated the ability of KLK6, 13, and 14 to knock down the protection against DSG-1 or Pro-FLG cleavage (fig. 5 c). Overall, these observations indicate that the ability of staphylococcus aureus to increase keratinocyte proteolytic activity by inducing KLK6, 13, and 14 can result in the digestion of molecules necessary to maintain a normal epidermal barrier.

Example 2

And (4) preparing bacteria. All bacteria used in this study are listed in table a. All staphylococcal strains (staphylococcus aureus, staphylococcus epidermidis, staphylococcus hominis, staphylococcus wowensis, staphylococcus capitis and staphylococcus lugdunensis) were grown to stationary phase in 3% Tryptic Soy Broth (TSB) at 250RPM in a 37 ℃ incubator at 4mL or 400 μ L volume (depending on the assay) for 24 h. Specific strains were grown under antibiotic selection (shown in table S1) at the following concentrations: 5 μ g/mL Erm, 25 μ g/mL Lcm and 10 μ g/mL Cm. For the treatment of bacterial supernatants of human keratinocytes or murine skin, 24h of cultured bacteria were pelleted (15min,4,000RPM, RT), and the supernatants were filter sterilized (0.22 μm). For murine and human keratinocyte assays with strains of human Staphylococcus C5 and Staphylococcus epidermidis RP62A, bacteria were filtered using a 3kDa size exclusion column (Amicon Ultra-15 centrifugal Filter, Millipore)Filtering the supernatant to collect<3kDa fraction and further concentrated 10X using a freeze dryer and then resuspended in molecular grade H before processing₂And (4) in O. Several techniques were used to further biochemically test human staphylococcal C5 supernatant. Ammonium sulfate precipitation (80%) was performed at room temperature for 1H, followed by centrifugation (30min,4,000RPM, RT), and then the precipitate (pellet) was washed in H₂Resuspend in O to isolate small peptides. In addition, the supernatant of human staphylococcal C5 was raised to pH 11 with 2M NaOH using a test strip at pH 1-14 for 1h, then 2M hcl was used to return the supernatant pH to the starting pH of about 6.5 before addition to the staphylococcus aureus agr reporter strain.

TABLE A

Culturing normal human keratinocyte. At 37 ℃ 5% CO₂Then, in the presence of 60. mu.M CaCl₂Normal fresh human epidermal keratinocytes (NHEK; Thermo Fisher Scientific) were cultured in Epilife's medium (Thermo Fisher Scientific) supplemented with 1x EpiLife's defined growth supplements (EDGS; Thermo Fisher Scientific) and 1x antibiotic-antimycotic (PSA; 100U/ml penicillin, 100U/ml streptomycin, 250ng/ml amphotericin B; Thermo Fisher Scientific). NHEK was used only for experiments between generations 3-5. For the experiment, NHEK was grown to 70% confluence and then in high calcium EpiLife medium (2mM CaCl)₂) Medium differentiation for 48 hours to mimic the upper layer of the epidermis. For bacterial supernatant treatment, differentiated NHEK was treated with sterile filtered bacterial supernatant (5% by volume) added to the Epilife medium for 24 h. Similar to the synthetic PSM treatment, 5-50. mu.g-mL of peptide was added to NHEK in DMSO for 24 h.

Staphylococcus aureus epidermal mouse model. As illustrated in the figure, 8 week old, sex and age matched male or female C57BL/6(Jackson) mice were used for all experiments (n-3-6). All Animal experiments were approved by the Institutional Animal Care and Use Committee. The mouse hair was removed by shaving and Nair was applied for 2-3min, then immediately removed with an alcohol wipe. The skin barrier was allowed to recover from hair removal for 48h prior to application of the bacteria. At 1.5cm²Staphylococcus aureus in 3% TSB (1e7 CFU) was applied to the skin of rats in 100. mu.L volumes on sterile gauze slides for 48-72 h. Tegaderm was applied on top of gauze to immobilize it during treatment. For the Staphylococcus aureus agr inhibition experiment, active human Staphylococcus C5(10:1) or 10X concentrated<The 3kDa sterile filtered commensal bacterial supernatant (1:1) was mixed with Staphylococcus aureus in 3% TSB and immediately applied to gauze.

Preparing the synthetic phenol soluble regulatory protein. All synthetic phenol soluble regulatory Proteins (PSMs) were produced by LifeTein (Hillsborough, NJ). The peptide with N-terminal formylation (f) was produced in 95% purity. The PSM sequence is as follows:

PSMα1:f-MGIIAGIIKVIKSLIEQFTGK(SEQ ID NO:5)、

PSMα2:f-MGIIAGIIKFIKGLIEKFTGK(SEQ ID NO:6)、

PSMα3:f-MEFVAKLFKFFKDLLGKFLGNN(SEQ ID NO:7)、

PSMα4:f-MAIVGTIIKIIKAIIDIFAK(SEQ ID NO:8)、

PSMβ2:

f-MTGLAEAIANTVQAAQQHDSVKLGTSIVDIVANGVGLLGKLFGF(SEQ ID NO:9)。

the peptides were resuspended in DMSO and concentrated by a vacuum centrifugal evaporator concentrator (speedvac) to 500mg of powdered starting material stored at-80 ℃ before reconstitution in DMSO for experiments.

RNA isolation and real-time quantitative PCR all RNAs were isolated using the Purelink RNA isolation kit (Thermo Fisher Scientific) according to the manufacturer's instructions for NHEK, 350. mu.L of RNA lysis buffer (containing 1% β -mercaptoethanol) was added directly to the columnAdded to the cells. For mouse tissues, 0.5cm in 750. mu.L of RNA lysis buffer²Full skin bead mill (2x 30sec,2.0mm zirconia beads) on ice for 5 minutes in the middle. The tissue was then centrifuged (10min, 13,000RPM, 4 ℃), and 350 μ Ι _ of the clarified lysate was added to 70% EtOH and column-based RNA isolation was performed. For S.aureus RNA isolation, 1X10⁹The CFU bacteria were incubated with RNAProtect (Qiagen) at a 2:1 ratio for 10min, then centrifuged (10min, 13,000RPM, RT), resuspended in 750. mu.L of RNA lysis buffer, and bead milled (2X1min, 6.5 speed) using lysis matrix B tubes and Fastprep-24(MP Biomedicals). The sample was then centrifuged again and 350 μ L of the clarified lysate was added to 70% EtOH as described above. After RNA isolation, samples were quantitated using a Nanodrop (ThermoFisher scientific) and 500ng RNA was reverse transcribed using the iScript cDNA Synthesis kit (Bio-Rad). The qPCR reaction was performed on a CFX96 real-time detection system (Bio-Rad). For mammalian cells, gene-specific primers and TaqMan probes (Thermo Fisher Scientific) were used, with GAPDH used as the housekeeping gene.

Generation and transformation of RP62A competent cells. Electrotransformation competent RP62A cells were prepared. Briefly, an overnight culture of Staphylococcus epidermidis RP62A was diluted to OD in pre-warmed Brain Heart Infusion (BHI) broth₆₀₀nm 0.5, incubated at 37 ℃ for a further 30min with shaking, transferred to centrifuge tubes and then frozen on ice for 10 min. Cells were harvested by centrifugation (10min, 4000RPM, 4 ℃), washed sequentially with 1 volume, 1/10 volumes, then 1/25 volumes of cold autoclaved water, and then re-pelleted at 4 ℃ after each wash. After the last wash, cells were resuspended in 1/200 volumes of cold 10% sterile glycerol and aliquoted into tubes at 50 μ Ι _ for storage at-80 ℃. Transformation of Staphylococcus epidermidis RP62A was performed. Briefly, frozen competent cells were thawed on ice for 5min and then at room temperature for 5 min. The thawed cells were briefly centrifuged (1min, 5000g, RT) and the pellet resuspended in 50 μ L of 10% glycerol supplemented with 500mM sucrose. After addition of DNA, cells were transferred to 1mm cups and at a time constant of 1.1msec, at 2.1kV, in MiPulses were performed on chopper (Bio-Rad). Immediately after electroporation, the cells were resuspended in 1mL BHI broth supplemented with 500mM sucrose, shaken at 30 ℃ for 1 hour, and then plated on BHI agar with 10. mu.g/mL chloramphenicol (Cm) at 30 ℃.

Allele substitution of Staphylococcus epidermidis RP62A AIP allele substitution plasmid pMAD (50) is used to selectively produce an in-frame deletion of the AIP coding sequence of agrD in Staphylococcus epidermidis RP 62A. fragments of approximately 1000bp upstream and downstream of the AIP sequence of RP62A are amplified by PCR and then ligated together by overlap extension or "SOEing" gene splicing the stitched fragments and pMAD vector are digested with BamHI and SalI, ligated together by T4 DNA ligase (NewEngland Biolabs) and subsequently used to chemically transform the Staphylococcus epidermidis colony 10 plasmid artificially modified E.coli strain DC 10B-CC10. the transformant is plated on LB with 100. mu.g/mL Amp and 30. mu.g/mLCm at 37 ℃ the correct transformant is verified by restriction digestion and verification of the transformant is verified as pMAD:: Δ P. B-10 and then incubated with a white agar medium with a cDNA encoding sequence of Gal-Gal cDNA encoding sequence from DC 10. 35. mu.g/mLCm.C.20. the white medium with a cDNA encoding sequence of the cDNA encoding sequence is plated in a white medium strain cDNA encoding strain 120. mu.g/mBHP strain III and incubated with agar medium for overnight growth of GalI-white medium-agar medium-white medium-agar medium-white medium-agar medium-white medium-agar medium-white medium-white medium-white medium-white medium-.

And (4) RNA sequencing. RNA was submitted to the university of california san diego branch school (UCSD) genomic core facility for library preparation and sequencing. Library preparation was performed using the TruSeq mRNA Library Prep Kit (TruSeq mRNA Library preparation Kit) (Illumina), followed by high throughput sequencing on a HiSeq 2500 sequencer (Illumina). Data were analyzed using the Partek Flow and Partek Genomics Suite software, and gene ontology analysis was performed using the PANTHER classification system (http [:/] pantherb.

Histology. Collecting the full thickness rat skin (0.5 cm)²) It was fixed in paraformaldehyde (4%) and washed in PBS, then incubated overnight with 30% and 10% sucrose, and then the tissue was frozen in OCT coverslips with dry ice. Cryostat cut sections (10mm) were mounted on Superfrost Plus slides (Fisher Scientific) and stained with hematoxylin and eosin (H)&E) And (6) dyeing. Sections were incubated with a 75% -100% gradient of EtOH for 5min intervals before xylene incubation and fixation with paramount and slides. Photographs were taken at 200x magnification on an Olympus BX51(Tokyo, Japan) fluorescence microscope.

Cytokine level assay conditioned media (25 μ L) from NHEK was used to quantify protein concentrations of various cytokines according to the manufacturer's instructions for the Magpix 200(Luminex) system, using a magnetic bead-based milliplex assay kit (Millipore) for 3 human cytokines (IL-6, IL-8, TNF α) human IL-1 α and IL-36 α were quantified by ELISA (R & DSystems).

Quantification of bacterial CFU. Staphylococcus aureus Colony Forming Units (CFU) were quantified as follows: in a 37 ℃ incubator, a 3% egg yolk emulsion and tellurite-containing Baird-parker agar (BD) plate was coated with 10^-1To 10^-5Serial dilutions of (10 μ L) were continued for 24h, and then CFU were counted. Bacterial CFU of all staphylococcus strains was estimated by using a spectrophotometer and also measuring the OD600nm of cells diluted 1:20 in PBS.

Transcutaneous water loss measurement. To determine damage to the epidermal skin barrier, the transdermal water loss (TEWL) was measured using TEWAMETER TM300(C & K) on mouse skin treated with staphylococcus aureus for 48-72 h.

Trypsin activity assay. NHEK conditioned medium was added at 50. mu.L to a black 96-well black plate (Corning), followed by 150. mu.L of the peptide Boc-Val-Pro-Arg-AMC (trypsin-like substrate; BACHEM) at a final concentration of 200. mu.M in 1 Xdigestion buffer (10mM Tris-HCl pH 7.8) and incubation at 37 ℃ for 24 h. Relative fluorescence intensities (ex:354nm, EM:435nm) were analyzed using a SpectraMAXGemini EM fluorometer (Thermo Fisher Scientific). For murine dermal trypsin activity assays, 0.5cm was incubated in 1mL of 1M acetic acid²Whole skin bead mill (2.0mm zirconia beads, 2x 30sec, 5min after each time) then spin at 4 ℃ overnight. The samples were centrifuged (10min, 13,000RPM, 4 ℃) and added to a new microcentrifuge tube, followed by protein concentration using Speedvac to remove any remaining acetic acid. The protein was resuspended in molecular-grade water (500. mu.L) and spun overnight at 4 ℃ before centrifugation again. The clarified protein lysate was added to a new tube and the protein concentration was determined using BCA (Bio-rad) analysis. Finally, 10 μ g of total protein was added to a 96-well plate and then analyzed with trypsin substrate as described above.

Staphylococcus aureus agr activity. Staphylococcus aureus agr activity was tested using Staphylococcus aureus USA300LAC agr type I P3-YFP (AH1677) or Staphylococcus aureus USA300LAC agr type I pAmi P3-Lux (AH2759) reporter strains. For in vitro experiments, 1e6CFU of Staphylococcus aureus USA300LAC agr type I P3-YFP was added to 300 μ L of 3% TSB together with 100 μ L of sterile filtered symbiotic supernatant (25% by volume) and shaken (250RPM) for 24h at 37 ℃. The bacteria were then diluted 1:20 in PBS (final concentration 200. mu.L) and YFP (ex:495nm, em:530nm) was detected as described above using a fluorimeter, followed by OD on a spectrophotometer₆₀₀The nm reading (readout) determines the bacterial density. For murine experiments, the activity of pAmiP3-Lux, type I LAC agr, Staphylococcus aureus USA300, was determined using the IVIS instrument, and the luminescence intensity after 2 minutes of exposure was evaluated by measuring the emitted photons (p/sec/cm2/sr) using the Liveimaging software (Perkinelmer).

Genome sequencing and assembly. Human staphylococcal C5 genomic DNA was isolated using DNeasy UltraClean microbiological Kit (Qiagen). The library was sequenced in two cycles using the MiSeq platform (Illumina inc., San Diego, CA) to generate paired-end reads of 2x250 bp. The linker was removed using cutatapt (version 1.9.1) (http [:/] cutatapt. Low quality sequences were removed with default parameters (quality score <30) using Trim Galore (version 1.9.1) (https [:// ] [ www. ] bioinformatics. babraham. ac. uk/projects/Trim _ Galore /). Sequences mapped to the human Genome were removed from the linker-depleted dataset using Bowtie2 (version 2.2.8) (51) and the human reference Genome hg19(UCSC Genome Browser) with the following parameters (-D20-R3-N1-L20- -very-sensitive-local). The filtered reads were assembled from scratch using SPAdes (version 3.8.0) (52), where the k-mer length ranged from 33 to 127. The genome was annotated with default parameters by rapid annotation of the microbial genome using subsystem technology (RAST). The amino acid sequence from the annotated CDS (coding DNA sequence) was aligned with the bacterial agr protein obtained from the Uniprot database (downloaded in 2017 at 10 months). The agr genes from the assembled genome were identified according to the following three criteria: i) sequence identity > 60%; ii) e value < e 100; and iii) agr locus structure, an operon of four genes, agrBDCA.

Microbiota data and comparative genomic analysis. Publicly available shotgun metagenomic data for atopic dermatitis skin was analyzed. The relative abundance of staphylococcus aureus and staphylococcus epidermidis strains was obtained directly from published supplementary materials ([ www. ] scientific transmethylation medicine. org/cgi/content/full/9/397/eal 4651/DC 1). agrD profiling was limited to eight patients (AD01, AD02, AD03, AD04, AD05, AD08, AD09, and AD11), with information on 7 different body sites on the red AD skin and differences in AD severity based on objective SCORAD. The 61 S.epidermidis strains evaluated were classified as agr I, II or III by amino acid sequence comparison with known agrD type I-III sequences in the agrD gene region.

Quantitative and statistical analysis. The nonparametric Mann-Whitney test was used for the statistical significance analysis of metagenomic data from AD patients. One-way analysis of variance or two-way analysis of variance for statistical analysis is indicated in the various graph descriptions. All statistical analyses were performed using GraphPad Prism version 6.0 (GraphPad, La Jolla, CA). All data are expressed as mean ± Standard Error of Mean (SEM) and P value ≦ 0.05 was considered significant.

The production of PSM α is essential for inducing trypsin-like serine protease activity and elevated mRNA levels of kallikrein-releasing enzyme 6(KLK6) (FIGS. 14A-B), PSM α and PSM 5390 in Staphylococcus aureus contain different peptides, including PSM α -4 and PSM 632-2, thus the expression of various proteins in the protein kinase map, PSM 6331-4, PSM-2, the protein kinase activity in NHPSM α, PSM-18-dependent protein kinase activity, PSM-9-2-PSM-7374, PSM-9-2-PSM-2-PSM-9-2-was found to affect the epidermal barrier activity in a wide spectrum of the expression of proteins (PSM-protein kinase-protein map) and PSM-protein kinase activity in the protein map-PSM-protein map-PSM-protein-PSM-map-protein-PSM-map-protein-was found to affect the epidermal barrier-protein synthesis in a wide spectrum-map-protein-.

To verify the effect of the PSM α operon on the epidermal barrier in vivo, the same number of staphylococcus aureus USA300LAC wild-type or PSM α mutant strains colonized 72h on the surface of mouse skin, desquamation (scaling) and epidermal thickening was induced by wild-type staphylococcus aureus, whereas no change in bacterial abundance was observed in the absence of PSM α (fig. 14F), despite the increase in epidermal thickness, an increase in transdermal water loss (TEWL) was observed after exposure to wild-type staphylococcus aureus, which is a mature method for assessing skin barrier damage, but this phenomenon was not observed in the absence of PSM α (fig. 14G), whereas the skin barrier disruption of the fully differentiated epidermis in vivo also depends on the expression of staphylococcus aureus proteases, the use of staphylococcus aureus 300LAC mutant strains lacking 10 secreted major proteases (including aureolysin, V8, staphopain a/B and SplA-F), although there are significant changes in the expression of staphylococcus aureus LAC 300LAC, which are measured in the presence of the major protease expression of the major proteases of aureolysin, which are responsible for the reduction in the epidermal barrier activity of the expression of these s 587 and the epidermal barrier proteins, which are measured in the environmental map of the increase of the expression of staphylococcus aureus, the epidermal barrier of the epidermal barrier-expressing the s-tr-map 15, the s-tr-map, the increase of the expression of the s-c promoter, the expression of the s-c mutant strains, which is measured in the expression of the s-c mutant strains, which is observed in the same as the expression of the map 15, which is observed in the map of the expression of the intact s-t map 16 s-tr-s, which is observed in the expression of the map, but which is observed in the same as the expression of the increase of the map, which is observed in the expression of the s, but which is observed in the expression.

Although there is increased skin colonization of staphylococcus aureus in AD, there are other bacterial species, such as coagulase-negative staphylococcus (cos) strains, including the abundant human skin commensal microorganism staphylococcus epidermidis, and it is therefore important to understand how these bacteria communicate, it has been demonstrated that staphylococcus epidermidis agr type I laboratory isolates are produced from An Inducer Peptide (AIP) which inhibits the staphylococcus aureus agr type I-III system by the cross-talk (crosstalk) mechanism of staphylococcus epidermidis, but not type IV, while the effects of other staphylococcus epidermidis agrII and type III on staphylococcus epidermidis agr activity are known to be less effective as inhibitors of staphylococcus epidermidis activity, when the activity of staphylococcus epidermidis induced from an inducer peptide inhibits staphylococcus epidermidis agr activity, staphylococcus epidermidis agi activity, or even is determined to be affected by the absence of the wild staphylococcus epidermidis agr protein kinase activity of staphylococcus epidermidis strain, when the staphylococcus epidermidis agr activity of staphylococcus epidermidis strain is tested for its lack of inhibition of staphylococcus epidermidis activity by wild staphylococcus epidermidis agp, staphylococcus epidermidis agp induction of staphylococcus epidermidis agp, staphylococcus epidermidis agii, staphylococcus epidermidis agp induction of staphylococcus epidermidis induced activity, staphylococcus epidermidis induced by wild staphylococcus epidermidis staphylococcus aureus agr agp, staphylococcus epidermidis induction of staphylococcus epidermidis, staphylococcus epidermidis agp, staphylococcus epidermidis induced by wild staphylococcus epidermidis induction of staphylococcus epidermidis, staphylococcus epidermidis agp, staphylococcus epidermidis induction of staphylococcus epidermidis agp, staphylococcus epidermidis induced staphylococcus epidermidis agp, staphylococcus epidermidis induced by staphylococcus epidermidis induced by staphylococcus epidermidis.

Lack of relative abundance of staphylococcus epidermidis agr type I on AD skin. Having determined the potential of laboratory strains of staphylococcus epidermidis to affect the effects of staphylococcus aureus on human keratinocyte function, experiments were conducted to determine the abundance of these bacteria in a clinical setting. Metagenomic data obtained from the skin microbiome collected from 7 body sites of 8 subjects with AD of varying severity (objective SCORAD-based) was analyzed for agr-type based relative abundance of staphylococcus epidermidis. Sequence alignment identified the staphylococcus epidermidis genome based on agr IIII type on AD patients and determined that the most common staphylococcus epidermidis agr type on AD skin was agr type I (fig. 15E). Comparison of staphylococcus epidermidis agr I with staphylococcus aureus showed that the abundance of staphylococcus epidermidis agr I-type became relatively small in AD subjects with increased disease severity (fig. 15F-G). These observations confirm the presence of staphylococcus epidermidis agr type I in the AD skin microbiota and suggest the possibility of association with clinical disease.

Different staphylococcal species and strains inhibit staphylococcus aureus agr activity. To further establish the physiological significance of the quorum sensing interaction between staphylococcus aureus and other members of the skin microbiome, culture supernatants of different AD clinical isolates of cos were tested for their ability to inhibit the quorum sensing activity of staphylococcus aureus USA300LAC agr type I. Different species (including staphylococcus epidermidis, staphylococcus hominis, staphylococcus wowensis and staphylococcus capitis) showed potent inhibitory activity against agr activity of staphylococcus aureus (fig. 16A). Similar to the laboratory isolates of staphylococcus epidermidis, the CoNS strain inhibited the agr activity of staphylococcus aureus, but not its growth rate (FIG. 316S). Furthermore, genomic sequence analysis of the agrD AIP coding region of human Staphylococcus strain C5 showed the presence of a novel AIP sequence in the AIP coding region, similar to that of the Staphylococcus epidermidis agr type I coding region, and with the predicted octamer AIP sequence of human Staphylococcus C5 (FIG. 16B; SEQ ID NO: 4). Biochemical techniques of the supernatant of active human Staphylococcus C5 showed that the inhibition of Staphylococcus aureus agr activity was dependent on a sensitive (thiolactone ring) factor at pH 11 of <3kDa (small size), which can be precipitated with 80% ammonium sulphate (peptide). (FIG. 16C).

Next, staphylococcus aureus was cultured in the presence of sterile filtered supernatant of staphylococcus hominis C5 and the subsequent culture supernatant was applied to NHEK as shown in fig. 14. Similar to staphylococcus epidermidis agr type I, human staphylococcus C5 inhibited staphylococcus aureus-induced trypsin activity, KLK6 transcript production, and IL-6 protein expression in NHEK (fig. 16D-F). Furthermore, in addition to the most common clinical isolates of agr type I, human staphylococcus C5 inhibited a variety of staphylococcus aureus agr systems, including agr type II and III, but not including type IV (fig. 17S). This finding is in agreement with that observed by the Staphylococcus epidermidis agr type I system. Collectively, these observations suggest that, in addition to staphylococcus epidermidis, clinical isolates of the CoNS species can also use quorum sensing to inhibit damage to keratinocytes by staphylococcus aureus.

Clinical CoNS isolates inhibited Staphylococcus aureus agr activity, its ability to promote AD. To establish a physiological correlation of quorum-sensing interactions between CoNS and Staphylococcus aureus in vivo, Staphylococcus aureus agr activity was evaluated by IVIS using a Staphylococcus aureus USA300LAC agr type I P3-Lux promoter (light-emitting) strain. Staphylococcus aureus on the back skin showed abundant agr activity, but when active human staphylococcus C5 was present, the agr activity of staphylococcus aureus was inhibited (fig. 17A-B). In addition, human staphylococcus C5 also protected against erythema and desquamation of skin caused by staphylococcus aureus (fig. 17C), without altering the abundance of staphylococcus aureus (fig. 17D). This phenotype was associated with inflammation, barrier disruption, and evidence of improvement in epidermal protease activity and Klk6 expression (fig. 17E-H). Furthermore, when staphylococcus aureus was applied to the skin of the rat back in the presence of a <3kDa concentrated supernatant of human staphylococcus C5, a similar reduction in barrier damage and inflammation was observed, while the abundance of staphylococcus aureus was unchanged. (FIG. 22). These data suggest that the skin CoNS microflora may contain novel AIPs that promote epithelial barrier homeostasis through interspecies quorum sensing activity.

Various embodiments of the present disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the disclosure. Accordingly, other embodiments are within the scope of the following claims.

Sequence listing

<110> board of president of california university

<120> molecular bacterial therapy for controlling the activity of dermal enzymes

<130>00015-332WO1

<140> has not specified yet

<141>2018-08-31

<150>US 62/553,025

<151>2017-08-31

<160>17

<170> PatentIn version 3.5

<210>1

<211>141

<212>DNA

<213> Staphylococcus human

<220>

<221>CDS

<222>(1)..(141)

<400>1

atg aca ttt att aca gat tta ttc att aaa cta ttc tca tta atc tta 48

Met Thr Phe Ile Thr Asp Leu Phe Ile Lys Leu Phe Ser Leu Ile Leu

1 5 10 15

gaa act gtt ggt aca ctt gct tca tat aat gta tgt ggt ggt tat ttc 96

Glu Thr Val Gly Thr Leu Ala Ser Tyr Asn Val Cys Gly Gly Tyr Phe

20 25 30

gat gaa cct gaa gtt cct aaa gaa tta act gat ctt aat aga taa 141

Asp Glu Pro Glu Val Pro Lys Glu Leu Thr Asp Leu Asn Arg

35 40 45

<210>2

<211>46

<212>PRT

<213> Staphylococcus human

<400>2

Met Thr Phe Ile Thr Asp Leu Phe Ile Lys Leu Phe Ser Leu Ile Leu

1 5 10 15

Glu Thr Val Gly Thr Leu Ala Ser Tyr Asn Val Cys Gly Gly Tyr Phe

20 25 30

Asp Glu Pro Glu Val Pro Lys Glu Leu Thr Asp Leu Asn Arg

35 40 45

<210>3

<211>27

<212>DNA

<213> Artificial sequence

<220>

<223> coding fragment from human Staphylococcus

<220>

<221>CDS

<222>(1)..(27)

<400>3

tca tat aat gta tgt ggt ggt tat ttc 27

Ser Tyr Asn Val Cys Gly Gly Tyr Phe

1 5

<210>4

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> Synthesis Structure

<400>4

Ser Tyr Asn Val Cys Gly Gly Tyr Phe

1 5

<210>5

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> phenol soluble regulatory protein α -1

<400>5

Met Gly Ile Ile Ala Gly Ile Ile Lys Val Ile Lys Ser Leu Ile Glu

1 5 10 15

Gln Phe Thr Gly Lys

20

<210>6

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> phenol soluble regulatory protein α -2

<400>6

Met Gly Ile Ile Ala Gly Ile Ile Lys Phe Ile Lys Gly Leu Ile Glu

1 5 10 15

Lys Phe Thr Gly Lys

20

<210>7

<211>22

<212>PRT

<213> Artificial sequence

<220>

<223> phenol soluble regulatory protein α -3

<400>7

Met Glu Phe Val Ala Lys Leu Phe Lys Phe Phe Lys Asp Leu Leu Gly

1 5 10 15

Lys Phe Leu Gly Asn Asn

20

<210>8

<211>20

<212>PRT

<213> Artificial sequence

<220>

<223> phenol soluble regulatory protein α -4

<400>8

Met Ala Ile Val Gly Thr Ile Ile Lys Ile Ile Lys Ala Ile Ile Asp

1 5 10 15

Ile Phe Ala Lys

20

<210>9

<211>44

<212>PRT

<213> Artificial sequence

<220>

<223> phenol soluble regulatory protein β -2

<400>9

Met Thr Gly Leu Ala Glu Ala Ile Ala Asn Thr Val Gln Ala Ala Gln

1 5 10 15

Gln His Asp Ser Val Lys Leu Gly Thr Ser Ile Val Asp Ile Val Ala

20 25 30

Asn Gly Val Gly Leu Leu Gly Lys Leu Phe Gly Phe

35 40

<210>10

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> AIP consensus sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223> Xaa is S, K, V, G or T

<220>

<221>MISC_FEATURE

<222>(2)..(2)

<223> Xaa is Y, Q, A or I

<220>

<221>MISC_FEATURE

<222>(3)..(3)

<223> Xaa is N, S, T or D

<220>

<221>MISC_FEATURE

<222>(4)..(4)

<223> Xaa is V, P, M or T

<220>

<221>MISC_FEATURE

<222>(6)..(6)

<223> Xaa is G, S, A, N or T

<220>

<221>MISC_FEATURE

<222>(7)..(7)

<223> Xaa is G, N, T or L

<220>

<221>MISC_FEATURE

<222>(8)..(8)

<223> Xaa is Y or F

<220>

<221>MISC_FEATURE

<222>(9)..(9)

<223> Xaa is Y, F or L

<400>10

Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa

1 5

<210>11

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> AIP peptide derived from Staphylococcus epidermidis strain A11

<400>11

Lys Tyr Asn Pro Cys Ser Asn Tyr Leu

1 5

<210>12

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> AIP peptides from human Staphylococcus strains A9 and C4

<400>12

Ser Tyr Ser Pro Cys Ala Thr Tyr Phe

1 5

<210>13

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> AIP peptide derived from human Staphylococcus

<400>13

Ser Gln Thr Val Cys Ser Gly Tyr Phe

1 5

<210>14

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> AIP peptides derived from human Staphylococcus and Staphylococcus capitis

<400>14

Gly Ala Asn Pro Cys Ala Leu Tyr Tyr

1 5

<210>15

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> AIP peptide derived from human Staphylococcus

<400>15

Thr Ile Asn Thr Cys Gly Gly Tyr Phe

1 5

<210>16

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> AIP peptides from Staphylococcus lugdunensis

<400>16

Val Gln Asp Met Cys Asn Gly Tyr Phe

1 5

<210>17

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> AIP peptide from Staphylococcus Wavorans strain G2

<400>17

Gly Tyr Ser Pro Cys Thr Asn Phe Phe

1 5

PCT/RO/134 Table

Claims

1. A purified polypeptide comprising a sequence having at least 98% identity to SEQ ID No. 4, 11, 12, 13, 14, 15, 16, or 17 and which inhibits (i) protease production and/or activity, (ii) IL-6 production and/or activity, (iii) production of phenol soluble regulatory protein α 3 by s.aureus, and/or (iv) agr production and/or activity by s.aureus.

2. The purified polypeptide of claim 1, wherein the polypeptide has at least 98% identity to SEQ ID NO 2.

3. The purified polypeptide of claim 1, wherein the polypeptide comprises SEQ ID NO 4, 11, 12, 13, 14, 15, 16, or 17.

4. The purified polypeptide of claim 1, wherein the polypeptide consists of SEQ ID NO 4, 11, 12, 13, 14, 15, 16, or 17.

5. The purified polypeptide of any of claims 1-4, wherein the polypeptide comprises one or more D-amino acids.

6. The purified polypeptide of any one of claims 1-4, wherein the polypeptide comprises a compound of formula I, formula IA, or formula IB.

7. A topical formulation comprising the polypeptide of any one of claims 1-4.

8. An isolated polynucleotide encoding the polypeptide of any one of claims 1-4.

9. The isolated polynucleotide of claim 8, wherein the polynucleotide comprises a sequence that hybridizes under stringent conditions to a polynucleotide consisting of SEQ ID No. 1 and encodes a polypeptide comprising SEQ ID No. 4.

10. The isolated polynucleotide of claim 8, wherein the polynucleotide comprises SEQ ID NO 1 or 3.

11. A vector comprising the polynucleotide of claim 8.

12. A vector comprising the polynucleotide of claim 9 or 10.

13. A recombinant microorganism comprising the polynucleotide of claim 8.

14. A recombinant microorganism comprising the polynucleotide of claim 9 or 10.

15. The recombinant microorganism of claim 13, wherein said microorganism is an attenuated microorganism.

16. The recombinant microorganism of claim 13, wherein said microorganism is a commensal microorganism.

17. A topical probiotic composition comprising the recombinant microorganism of claim 15 or 16.

18. A topical probiotic composition consisting of a microorganism expressing the polypeptide of claim 1.

19. The topical probiotic composition of claim 18, wherein said microorganism is staphylococcus hominis (s.hominis), staphylococcus epidermidis (s.epidermidis), staphylococcus wowensis (s.wanneri), or any combination thereof.

20. A topical probiotic composition according to claim 19, wherein said microorganism is staphylococcus hominis C5, staphylococcus hominis a9, staphylococcus epidermidis a11 and/or staphylococcus wavorans G2.

21. The topical probiotic composition according to claim 18, wherein said composition comprises a microorganism selected from the group of microorganisms consisting of: the microorganism has ATCC No. _______ (strain name staphylococcus epidermidis a 1181618, deposited 28 months at 2018), ATCC No. _______ (strain name staphylococcus epidermidis C581618, deposited 28 months at 2018 months 28 days at 2018), ATCC No. _______ (strain name staphylococcus epidermidis a981618, deposited 28 months at 2018 months 28 days at 2018), ATCC No. _______ (strain name staphylococcus wadskii G281618, deposited 28 months at 2018 months 8 days) and combinations of any of the foregoing.

22. A method of treating a dermatological disorder, comprising administering an effective amount of a coagulase-negative Staphylococcus species (suns sp.) (cos) or an effective amount of a fermented extract of cos, sufficient to inhibit protease activity on skin, wherein the cos produces a polypeptide comprising a sequence at least 98% identical to SEQ ID No. 4 and inhibits protease production.

23. The method of claim 22, wherein the dermatological disorder is selected from the group consisting of: netherton syndrome, atopic dermatitis, contact dermatitis, eczema, psoriasis, acne, epidermal hyperkeratosis, acanthosis, epidermal inflammation, dermal inflammation and pruritus.

24. The method of claim 22, wherein said administering is by topical application.

25. The method of claim 22, wherein the CoNS is selected from the group consisting of: staphylococcus epidermidis (Staphylococcus epidermidis), Staphylococcus capitis (Staphylococcus capitis), Staphylococcus caprae (Staphylococcus caprae), Staphylococcus saccharolyticus (Staphylococcus saccharolyticus), Staphylococcus wowen (Staphylococcus warneri), Staphylococcus pasteuri (Staphylococcus pasteurii), Staphylococcus haemolyticus (Staphylococcus haemolyticus), Staphylococcus delbrueckii (Staphylococcus devrieii), Staphylococcus Hominis (Staphylococcus aureus mini), Staphylococcus aureus, and Staphylococcus lugdunensis (Staphylococcus lugdunensis).

26. The method of claim 22, wherein the fermented extract of CoNS comprises the polypeptide sequence of SEQ ID NO 4 and/or the compound of formula I.

27. The method of claim 22, wherein the CoNS is selected from the group consisting of: staphylococcus epidermidis a11, staphylococcus hominis C4, staphylococcus hominis C5, staphylococcus hominis a9, staphylococcus woolli G2 and any combination thereof.

28. A method of treating a skin disease or condition, the method comprising:

measuring protease activity of a culture from the skin of the subject or protease activity of the skin from the subject;

comparing the protease activity to a normal control;

applying a symbiotic skin bacteria composition and/or a fermented extract from coagulase-negative staphylococci, wherein the symbiotic skin bacteria composition or fermented extract comprises a polypeptide having at least 98% identity to SEQ ID No. 4 and/or comprises a compound of formula I, wherein the composition is formulated as a cream, ointment or pharmaceutical composition that maintains the growth and replication capacity of the symbiotic skin bacteria.

29. The method of claim 28, wherein the coagulase-negative staphylococci is selected from the group consisting of: staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus saccharolyticus, Staphylococcus wowensis, Staphylococcus pasteuri, Staphylococcus haemolyticus, Staphylococcus delbrueckii, Staphylococcus hominis, Staphylococcus aureus jettentis, Staphylococcus petasii and Staphylococcus lugdunensis.

30. A method of treating a skin disease or disorder comprising administering the purified polypeptide of claim 1 or a probiotic composition comprising a bacterium that produces a polypeptide having at least 95% identity to SEQ ID No. 4 that inhibits the expression of kallikrein.

31. A method of treating a skin disease or disorder comprising administering a composition that inhibits expression of a phenol soluble regulatory protein, wherein the composition comprises the purified polypeptide of claim 1 or the compound of formula I.

32. The method of claim 30 or 31, wherein the administration is topical administration.

33. The method of claim 30 or 31, wherein the composition is a fermented extract of coagulase-negative staphylococci.

34. A topical probiotic composition comprising a plurality of probiotic commensal skin bacteria selected from the group consisting of: staphylococcus epidermidis a11, staphylococcus hominis C4, staphylococcus hominis C5, staphylococcus hominis a9, staphylococcus woolli G2 and any combination thereof.

35. The topical probiotic composition of claim 34, formulated as a lotion, shake, cream, ointment, gel, foam, powder, solid, paste, or tincture.

36. A pharmaceutical composition comprising a drug and a fermented extract of staphylococcus aureus or an organism of staphylococcus aureus comprising phenolic soluble regulatory protein α 3.

37. A method of drug delivery through the skin comprising contacting the skin with the composition of claim 36.